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TW202540200A - Anti-fap-light fusion protein and use thereof - Google Patents

Anti-fap-light fusion protein and use thereof

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Publication number
TW202540200A
TW202540200A TW113146439A TW113146439A TW202540200A TW 202540200 A TW202540200 A TW 202540200A TW 113146439 A TW113146439 A TW 113146439A TW 113146439 A TW113146439 A TW 113146439A TW 202540200 A TW202540200 A TW 202540200A
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Taiwan
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amino acid
acid sequence
seq
region
binding
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TW113146439A
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Chinese (zh)
Inventor
翟倩婷
費明健
曹俊
孫潤孜
李莉雪
朱慧
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美商基利科學股份有限公司
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Publication of TW202540200A publication Critical patent/TW202540200A/en

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Abstract

The present disclosure relates to an anti-FAP antibody or antigen-binding fragment thereof, a bispecific antibody, a fusion protein, pharmaceutical compositions, and the uses of such antibodies in preventing, diagnosing, or treating tumor diseases.

Description

抗FAP-LIGHT融合蛋白及其用途Anti-FAP-LIGHT fusion protein and its uses

本揭露係關於抗FAP抗體、融合蛋白、及其用途。This disclosure relates to anti-FAP antibodies, fusion proteins, and their uses.

本節中的陳述僅提供與本揭露相關的先前技術資訊，且不必然構成先前技術。The statements in this section provide prior art information in connection with this disclosure only and do not necessarily constitute prior art.

TNFSF14（TNF超家族成員14 (TNF superfamily member 14)），亦稱為LIGHT，係TNF超家族的成員，且與淋巴毒素同源。LIGHT係一種可誘導型炎性細胞介素，其結合至TNFRSF14（TNF受體超家族成員14 (TNF receptor superfamily member 14)，亦稱為HVEM）、LTβR（淋巴毒素β受體(lymphotoxin beta receptor)）、及誘餌受體DcR3。此外，其與單純疱疹病毒醣蛋白D競爭與HVEM受體的結合。HVEM在多種免疫細胞的表面上表現，諸如T細胞、B細胞、NK細胞、及樹突細胞。LIGHT結合至HVEM，且隨後刺激T細胞並促進發炎。另一受體LTβR在上皮、基質、未成熟DC、及其他骨髓細胞的表面上表現，但不在淋巴細胞上表現。LTβR信號傳導之活化對於免疫細胞之募集及組織至關重要，導致淋巴器官及三級淋巴結構(TLS)之發育。TLS由B細胞及T細胞區、樹突細胞、及其他免疫細胞組成，且其等在腫瘤組織中的存在與多種癌症適應症（包括乳癌、肺癌、及結腸癌）中臨床結果的改善相關聯，這表明TLS可能在針對癌症的免疫反應中發揮重要作用。TNFSF14 (TNF superfamily member 14), also known as LIGHT, is a member of the TNF superfamily and is homologous to lymphotoxin. LIGHT is an inducible inflammatory cytokine that binds to TNFRSF14 (TNF receptor superfamily member 14, also known as HVEM), LTβR (lymphotoxin beta receptor), and the pheromone receptor DcR3. Furthermore, it competes with herpes simplex virus glycoprotein D for binding to the HVEM receptor. HVEM is expressed on the surface of various immune cells, such as T cells, B cells, NK cells, and dendritic cells. LIGHT binds to HVEM and subsequently stimulates T cells and promotes inflammation. Another receptor, LTβR, is expressed on the surface of epithelial cells, stromal cells, immature dendritic cells (DCs), and other bone marrow cells, but not on lymphocytes. Activation of LTβR signaling is crucial for the recruitment and organization of immune cells, leading to the development of lymphoid organs and tertiary lymphoid structures (TLS). TLS consists of B cell and T cell regions, dendritic cells, and other immune cells, and their presence in tumor tissue is associated with improved clinical outcomes in various cancer indications, including breast, lung, and colon cancer, suggesting that TLS may play an important role in the immune response against cancer.

本文提供一種經單離抗FAP抗體或其FAP抗原結合片段，其包含： a) i)     重鏈可變區(VH)，其包含 (1)  包含IYGVN (SEQ ID NO: 26)之胺基酸序列的重鏈互補決定區1 (HCDR1)， (2)  包含AIWSGGRKDYX 2LSLKS (SEQ ID NO: 27)之胺基酸序列的重鏈互補決定區2 (HCDR2)，其中X 2係N或S，及 (3)  包含SQDMPGYFDY (SEQ ID NO: 28)之胺基酸序列的重鏈互補決定區3 (HCDR3)，及 ii)    輕鏈可變區(VL)，其包含 (1)  包含KTNQNVDYX 1GNTFMH (SEQ ID NO: 23)之胺基酸序列的輕鏈互補決定區1 (LCDR1)，其中X 1係N或S， (2)  包含LASNLAS (SEQ ID NO: 24)之胺基酸序列的輕鏈互補決定區2 (LCDR2)，及 (3)  包含QQSRNLPYT (SEQ ID NO: 25)之胺基酸序列的輕鏈互補決定區3 (LCDR3)； 或 b) i)     VH區，其包含 (1)  包含TAGMSVG (SEQ ID NO: 32)之胺基酸序列的HCDR1， (2)  包含DIWWDDKKHYNPSLKD (SEQ ID NO: 33)之胺基酸序列的HCDR2， (3)  包含DMIFNFYFDV (SEQ ID NO: 34)之胺基酸序列的HCDR3，及 ii)    VL區，其包含 (1)  包含SASSRVGYMH (SEQ ID NO: 29)之胺基酸序列的LCDR1， (2)  包含DTSKLAS (SEQ ID NO: 30)之胺基酸序列的LCDR2，及 (3)  包含FQGSGYPFT (SEQ ID NO: 31)之胺基酸序列的LCDR3。 This article provides a single-chain anti-FAP antibody or its FAP antigen-binding fragment, comprising: a) i) a heavy chain variable region (VH) comprising (1) a heavy chain complement determination region 1 (HCDR1) comprising an amino acid sequence of IYGVN (SEQ ID NO: 26), (2) a heavy chain complement determination region 2 (HCDR2) comprising an amino acid sequence of AIWSGGRKDYX 2 LSLKS (SEQ ID NO: 27), wherein X 2 is N or S, and (3) a heavy chain complement determination region 3 (HCDR3) comprising an amino acid sequence of SQDMPGYFDY (SEQ ID NO: 28), and ii) a light chain variable region (VL) comprising (1) a light chain variable region comprising KTNQNVDYX 1 GNTFMH (SEQ ID NO: 28), wherein X 2 is N or S, and (2) a heavy chain complement determination region 3 (HCDR3) comprising an amino acid sequence of AIWSGGRKDYX 2 LSLKS (SEQ ID NO: 28), and (3) a light chain variable region (VL) comprising (1) a light chain variable region comprising KTNQNVDYX 1 GNTFMH (SEQ ID NO: 28), wherein X 2 is N or S, and (4) a light chain variable region comprising KTNQNVDYX 1 GNTFMH (SEQ ID NO: 28), wherein X 2 is N or S, and (5) a light chain variable region comprising KTNQNVDYX 1 GNTFMH (SEQ ID NO: 28), wherein X 2 is N or S, and (6) a light chain variable region comprising KTNQNVDYX 1 GNTFMH (SEQ ID NO: 28), wherein X 2 is N or S, and (7) a light chain variable region comprising KTNQNVDYX 1 GNTFMH (SEQ ID NO: 28), wherein X 2 is N or S, and (8) a light chain variable region comprising KTNQNVDYX 1 GNTFMH (SEQ ID 23) Light chain complementarity determining region 1 (LCDR1) of the amino acid sequence, wherein X1 is N or S, (2) Light chain complementarity determining region 2 (LCDR2) of the amino acid sequence of LASSNLAS (SEQ ID NO: 24), and (3) Light chain complementarity determining region 3 (LCDR3) of the amino acid sequence of QQSRNLPYT (SEQ ID NO: 25); or b) i) VH region, which includes (1) HCDR1 of the amino acid sequence of TAGMSVG (SEQ ID NO: 32), (2) HCDR2 of the amino acid sequence of DIWWDDKKHYNPSLKD (SEQ ID NO: 33), (3) HCDR3 of the amino acid sequence of DMIFNFYFDV (SEQ ID NO: 34), and ii) VL region, which includes (1) LCDR1 containing the amino acid sequence SASSRVGYMH (SEQ ID NO: 29), LCDR2 containing the amino acid sequence DTSKLAS (SEQ ID NO: 30), and LCDR3 containing the amino acid sequence FQGSGYPFT (SEQ ID NO: 31).

在一些實施例中，經單離抗FAP抗體或其FAP抗原結合片段包含： a)    VH區，其包含與如SEQ ID NO: 35所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；及/或VL區，其包含與如SEQ ID NO: 36所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列； b)   VH區，其包含與如SEQ ID NO: 37所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；及/或VL區，其包含與如SEQ ID NO: 38所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；或 c)    VH區，其包含與如SEQ ID NO: 41所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；及/或VL區，其包含與如SEQ ID NO: 42所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列。 In some embodiments, the isolated FAP antibody or its FAP antigen-binding fragment comprises: a) a VH region containing an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identical to or the same as the amino acid sequence shown in SEQ ID NO: 35; and/or a VL region containing an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identical to or the same as the amino acid sequence shown in SEQ ID NO: 36; b) a VH region containing an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identical to or the same as the amino acid sequence shown in SEQ ID NO: 36; The amino acid sequence shown in SEQ ID NO: 37 contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 38; and/or the VL region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 38; or c) the VH region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 41; and/or the VL region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 38; The amino acid sequence shown in 42 contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or an amino acid sequence identical to it.

在一些實施例中，經單離抗FAP抗體或其FAP抗原結合片段包含： a)    重鏈(HC)區，其包含與如SEQ ID NO: 1、4、5、6、8、9、10、11、12、13、14、15、及16所示之胺基酸序列中之任一者包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；及/或 b)   輕鏈(LC)區，其包含與如SEQ ID NO: 2、3、及7所示之胺基酸序列中之任一者包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列。 In some embodiments, the isolated FAP antibody or its FAP antigen-binding fragment comprises: a) a heavy chain (HC) region comprising an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence-identical to or identical with any of the amino acid sequences shown in SEQ ID NO: 1, 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, and 16; and/or b) a light chain (LC) region comprising an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence-identical to or identical with any of the amino acid sequences shown in SEQ ID NO: 2, 3, and 7.

在一些實施例中，經單離抗FAP抗體或其FAP抗原結合片段包含： a)    HC區，其包含與如SEQ ID NO: 1所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；及/或LC區，其包含與如SEQ ID NO: 2所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；或 b)   HC區，其包含與如SEQ ID NO: 4所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；及/或LC區，其包含與如SEQ ID NO: 3所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列； c)    第一重鏈(HC1)區，其包含與如SEQ ID NO: 5所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；第二重鏈(HC2)區，其包含與如SEQ ID NO: 6所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；及/或LC區，其包含與如SEQ ID NO: 3所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列； d)   HC1區，其包含與如SEQ ID NO: 8所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；HC2區，其包含與如SEQ ID NO: 9所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；及/或LC區，其包含與如SEQ ID NO: 7所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列； e)    HC1區，其包含與如SEQ ID NO: 10所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；HC2區，其包含與如SEQ ID NO: 11所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；及/或LC區，其包含與如SEQ ID NO: 3所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列； f)    HC1區，其包含與如SEQ ID NO: 13所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；HC2區，其包含與如SEQ ID NO: 12所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；及/或LC區，其包含與如SEQ ID NO: 3所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列； g)   HC1區，其包含與如SEQ ID NO: 14所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；HC2區，其包含與如SEQ ID NO: 13所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；及/或LC區，其包含與如SEQ ID NO: 3所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；或 h)   HC1區，其包含與如SEQ ID NO: 15所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；HC2區，其包含與如SEQ ID NO: 16所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；及/或LC區，其包含與如SEQ ID NO: 3所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列。 In some embodiments, the isolated FAP antibody or its FAP antigen-binding fragment comprises: a) an HC region comprising an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to or the same as the amino acid sequence shown in SEQ ID NO: 1; and/or an LC region comprising an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to or the same as the amino acid sequence shown in SEQ ID NO: 2; or b) an HC region comprising an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to or the same as the amino acid sequence shown in SEQ ID NO: 2; The amino acid sequence shown in SEQ ID NO: 4 contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 3; and/or the LC region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 5; c) The first heavy chain (HC1) region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 5; the second heavy chain (HC2) region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 5; The amino acid sequence shown in SEQ ID NO: 6 contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 3; and/or the LC region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 3; d) The HC1 region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 8; the HC2 region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 8; The amino acid sequence shown in SEQ ID NO: 9 contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 7; and/or the LC region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 7; e) the HC1 region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 10; the HC2 region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 10; The amino acid sequence shown in SEQ ID NO: 11 contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 3; and/or the LC region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 3; f) The HC1 region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 13; the HC2 region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 13; The amino acid sequence shown in SEQ ID NO: 12 contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 3; and/or the LC region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 3; g) The HC1 region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 14; the HC2 region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 14; The amino acid sequence shown in SEQ ID NO: 13 contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 3; and/or the LC region, which contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 3; or h) the HC1 region, which contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 15; the HC2 region, which contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 15; The amino acid sequence shown in SEQ ID NO: 3 contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical amino acid sequence; and/or the LC region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical amino acid sequence as shown in SEQ ID NO: 3.

在一些實施例中，VH區及/或VL區進一步包含人類架構序列。在一些實施例中，VH區及/或VL區進一步包含架構1 (FR1)、架構2 (FR2)、架構3 (FR3)、及/或架構4 (FR4)序列。在一些實施例中，本文所提供之經單離抗FAP抗體或FAP抗原結合片段包含衍生自免疫球蛋白之可結晶片段(Fc)區。在一些實施例中，Fc片段係衍生自IgG1、IgG2、IgG3、或IgG4，可選地，Fc片段係衍生自IgG4。在一些實施例中，Fc片段包含突變S228P或LALAPG突變。In some embodiments, the VH and/or VL regions further comprise human structural sequences. In some embodiments, the VH and/or VL regions further comprise structure 1 (FR1), structure 2 (FR2), structure 3 (FR3), and/or structure 4 (FR4) sequences. In some embodiments, the monoclonal anti-FAP antibody or FAP antigen-binding fragment provided herein comprises a crystallizable fragment (Fc) region derived from an immunoglobulin. In some embodiments, the Fc fragment is derived from IgG1, IgG2, IgG3, or IgG4; alternatively, the Fc fragment is derived from IgG4. In some embodiments, the Fc fragment comprises the S228P or LALAPG mutation.

在一些實施例中，抗體係單株抗體。在一些實施例中，抗體係人源化、人類、或嵌合抗體。在一些實施例中，抗FAP抗體或FAP抗原結合片段與人類、食蟹獼猴、及小鼠FAP交叉反應。在一些實施例中，抗體或其片段係Fab、Fab’、F(ab’)2、Fv、scFv、(scFv)2、單鏈抗體分子、雙可變區抗體、單可變區抗體、線性抗體、V區、或由抗體片段形成之多特異性抗體。在一些實施例中，經單離抗FAP抗體或其FAP抗原結合片段以不大於20 nM、15 nM、10 nM、或5 nM之解離常數(KD)結合至FAP。In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a humanized, human, or chimeric antibody. In some embodiments, the anti-FAP antibody or FAP antigen-binding fragment cross-reacts with human, cynomolgus monkey, and mouse FAP. In some embodiments, the antibody or fragment thereof is Fab, Fab’, F(ab’)2, Fv, scFv, (scFv)2, a single-chain antibody molecule, a bivariate antibody, a monovariate antibody, a linear antibody, a V-region antibody, or a multispecific antibody formed from an antibody fragment. In some embodiments, the isolated anti-FAP antibody or its FAP antigen-binding fragment binds to FAP with a dissociation constant (KD) not greater than 20 nM, 15 nM, 10 nM, or 5 nM.

本文亦提供一種雙特異性抗體或其抗原結合片段，其包含FAP抗原結合部份及第二結合部份，其中該FAP抗原結合部份包含： a) i)     重鏈可變區(VH)，其包含 (1)  包含IYGVN (SEQ ID NO: 26)之胺基酸序列的重鏈互補決定區1 (HCDR1)， (2)  包含AIWSGGRKDYX 2LSLKS (SEQ ID NO: 27)之胺基酸序列的重鏈互補決定區2 (HCDR2)，其中X 2係N或S，及 (3)  包含SQDMPGYFDY (SEQ ID NO: 28)之胺基酸序列的重鏈互補決定區3 (HCDR3)，及 ii)    輕鏈可變區(VL)，其包含 (1)  包含KTNQNVDYX 1GNTFMH (SEQ ID NO: 23)之胺基酸序列的輕鏈互補決定區1 (LCDR1)，其中X 1係N或S， (2)  包含LASNLAS (SEQ ID NO: 24)之胺基酸序列的輕鏈互補決定區2 (LCDR2)，及 (3)  包含QQSRNLPYT (SEQ ID NO: 25)之胺基酸序列的輕鏈互補決定區3 (LCDR3)； 或 b) i)     VH區，其包含 (1)  包含TAGMSVG (SEQ ID NO: 32)之胺基酸序列的HCDR1， (2)  包含DIWWDDKKHYNPSLKD (SEQ ID NO: 33)之胺基酸序列的HCDR2， (3)  包含DMIFNFYFDV (SEQ ID NO: 34)之胺基酸序列的HCDR3，及 ii)    VL區，其包含 (1)  包含SASSRVGYMH (SEQ ID NO: 29)之胺基酸序列的LCDR1， (2)  包含DTSKLAS (SEQ ID NO: 30)之胺基酸序列的LCDR2，及 (3)  包含FQGSGYPFT (SEQ ID NO: 31)之胺基酸序列的LCDR3。 This document also provides a bispecific antibody or its antigen-binding fragment thereof, comprising an FAP antigen-binding portion and a second binding portion, wherein the FAP antigen-binding portion comprises: a) i) a heavy chain variable region (VH), comprising (1) a heavy chain complementarity-determining region 1 (HCDR1) comprising an amino acid sequence of IYGVN (SEQ ID NO: 26), (2) a heavy chain complementarity-determining region 2 (HCDR2) comprising an amino acid sequence of AIWSGGRKDYX 2 LSLKS (SEQ ID NO: 27), wherein X 2 is N or S, and (3) a heavy chain complementarity-determining region 3 (HCDR3) comprising an amino acid sequence of SQDMPGYFDY (SEQ ID NO: 28), and ii) a light chain variable region (VL), comprising (1) The light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence KTNQNVDYX 1 GNTFMH (SEQ ID NO: 23), wherein X1 is N or S; (2) the light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence LASNLAS (SEQ ID NO: 24); and (3) the light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence QQSRNLPYT (SEQ ID NO: 25); or b) i) the VH region comprising (1) HCDR1 comprising the amino acid sequence TAGMSVG (SEQ ID NO: 32), (2) HCDR2 comprising the amino acid sequence DIWWDDKKHYNPSLKD (SEQ ID NO: 33), and (3) HCDR2 comprising the amino acid sequence DMIFNFYFDV (SEQ ID NO: 23). 34) HCDR3 of the amino acid sequence, and ii) VL region, which includes (1) LCDR1 containing the amino acid sequence SASSRVGYMH (SEQ ID NO: 29), (2) LCDR2 containing the amino acid sequence DTSKLAS (SEQ ID NO: 30), and (3) LCDR3 containing the amino acid sequence FQGSGYPFT (SEQ ID NO: 31).

在一些實施例中，FAP抗原結合部份包含： a)    VH區，其包含與如SEQ ID NO: 35所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；及/或VL區，其包含與如SEQ ID NO: 36所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列； b)   VH區，其包含與如SEQ ID NO: 37所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；及/或VL區，其包含與如SEQ ID NO: 38所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；或 c)    VH區，其包含與如SEQ ID NO: 41所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；及/或VL區，其包含與如SEQ ID NO: 42所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列。 In some embodiments, the FAP antigen-binding region comprises: a) a VH region containing an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with or identical to the amino acid sequence shown in SEQ ID NO: 35; and/or a VL region containing an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with or identical to the amino acid sequence shown in SEQ ID NO: 36; b) a VH region containing an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with or identical to the amino acid sequence shown in SEQ ID NO: 36; The amino acid sequence shown in SEQ ID NO: 37 contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 38; and/or the VL region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 38; or c) the VH region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 41; and/or the VL region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 38; The amino acid sequence shown in 42 contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or an amino acid sequence identical to it.

在一些實施例中，FAP抗原結合部份包含： c)    重鏈(HC)區，其包含與如SEQ ID NO: 1、4、5、6、8、9、10、11、12、13、14、15、及16所示之胺基酸序列中之任一者包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；及/或 d)   輕鏈(LC)區，其包含與如SEQ ID NO: 2、3、及7所示之胺基酸序列中之任一者包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列。 In some embodiments, the FAP antigen-binding region includes: c) a heavy chain (HC) region containing an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence-identical to or identical with any of the amino acid sequences shown in SEQ ID NO: 1, 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, and 16; and/or d) a light chain (LC) region containing an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence-identical to or identical with any of the amino acid sequences shown in SEQ ID NO: 2, 3, and 7.

在一些實施例中，FAP抗原結合部份包含： a)    HC區，其包含與如SEQ ID NO: 1所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；及/或LC區，其包含與如SEQ ID NO: 2所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；或 b)   HC區，其包含與如SEQ ID NO: 4所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；及/或LC區，其包含與如SEQ ID NO: 3所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列； c)    第一重鏈(HC1)區，其包含與如SEQ ID NO: 5所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；第二重鏈(HC2)區，其包含與如SEQ ID NO: 6所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；及/或LC區，其包含與如SEQ ID NO: 3所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列； d)   HC1區，其包含與如SEQ ID NO: 8所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；HC2區，其包含與如SEQ ID NO: 9所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；及/或LC區，其包含與如SEQ ID NO: 7所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列； e)    HC1區，其包含與如SEQ ID NO: 10所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；HC2區，其包含與如SEQ ID NO: 11所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；及/或LC區，其包含與如SEQ ID NO: 3所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列； f)    HC1區，其包含與如SEQ ID NO: 13所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；HC2區，其包含與如SEQ ID NO: 12所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；及/或LC區，其包含與如SEQ ID NO: 3所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列； g)   HC1區，其包含與如SEQ ID NO: 14所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；HC2區，其包含與如SEQ ID NO: 13所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；及/或LC區，其包含與如SEQ ID NO: 3所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；或 h)   HC1區，其包含與如SEQ ID NO: 15所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；HC2區，其包含與如SEQ ID NO: 16所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；及/或LC區，其包含與如SEQ ID NO: 3所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列。 In some embodiments, the FAP antigen-binding region includes: a) an HC region containing an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with or identical to the amino acid sequence shown in SEQ ID NO: 1; and/or an LC region containing an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with or identical to the amino acid sequence shown in SEQ ID NO: 2; or b) an HC region containing an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with or identical to the amino acid sequence shown in SEQ ID NO: 2; The amino acid sequence shown in SEQ ID NO: 4 contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 3; and/or the LC region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 5; c) The first heavy chain (HC1) region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 5; the second heavy chain (HC2) region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 5; The amino acid sequence shown in SEQ ID NO: 6 contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 3; and/or the LC region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 3; d) The HC1 region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 8; the HC2 region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 8; The amino acid sequence shown in SEQ ID NO: 9 contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 7; and/or the LC region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 7; e) the HC1 region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 10; the HC2 region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 10; The amino acid sequence shown in SEQ ID NO: 11 contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 3; and/or the LC region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 3; f) The HC1 region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 13; the HC2 region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 13; The amino acid sequence shown in SEQ ID NO: 12 contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 3; and/or the LC region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 3; g) The HC1 region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 14; the HC2 region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 14; The amino acid sequence shown in SEQ ID NO: 13 contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 3; and/or the LC region, which contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 3; or h) the HC1 region, which contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 15; the HC2 region, which contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 15; The amino acid sequence shown in SEQ ID NO: 3 contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical amino acid sequence; and/or the LC region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical amino acid sequence as shown in SEQ ID NO: 3.

在一些實施例中，雙特異性抗體或其抗原結合片段在FAP抗原結合部份之N端包含衍生自免疫球蛋白之Fc片段。在一些實施例中，Fc片段係衍生自IgG1、IgG2、IgG3、或IgG4，可選地，Fc片段係衍生自IgG4。在一些實施例中，Fc片段包含突變S228P或LALAPG突變。在一些實施例中，雙特異性抗體或其抗原結合片段包含Fc片段，且其中Fc片段包含選自由下列所組成之群組的一或多個修飾：鈕扣(knobs-into-holes)、DDKK、CH3之靜電轉向(electrostatic steering)、DuoBody、SEEDbodies、cFAE、XmAb、Azymetric、及BEAT ®，可選地，Fc片段包含修飾鈕扣及/或DDKK。 In some embodiments, the bispecific antibody or its antigen-binding fragment includes an Fc fragment derived from an immunoglobulin at the N-terminus of the FAP antigen-binding region. In some embodiments, the Fc fragment is derived from IgG1, IgG2, IgG3, or IgG4; alternatively, the Fc fragment is derived from IgG4. In some embodiments, the Fc fragment contains a mutant S228P or LALAPG mutation. In some embodiments, the bispecific antibody or its antigen-binding fragment includes an Fc fragment, wherein the Fc fragment contains one or more modifications selected from the group consisting of: knots-into-holes, DDKK, electrostatic steering of CH3, DuoBody, SEEDbodies, cFAE, XmAb, Azymetric, and BEAT® ; alternatively, the Fc fragment contains modified knots and/or DDKK.

在一些實施例中，第二結合部份係在FAP抗原結合部份之C端。在一些實施例中，第二結合部份係可操作地連接至Fc片段之C端。在一些實施例中，第二結合部份藉由連接子連接至Fc片段。在一些實施例中，第二結合部份結合及/或活化第二目標。在一些實施例中，第二結合部份結合及/或活化腫瘤相關細胞受體。在一些實施例中，第二結合部份包含第一部分及第二部分，其中各部分包含一或多個單元。在一些實施例中，一或多個單元包含第一單元、第二單元、及/或第三單元。在一些實施例中，一或多個單元之各者係相同的。在一些實施例中，一或多個單元係不相同的。在一些實施例中，第二結合部份係腫瘤壞死因子、介白素、淋巴激素、干擾素、群落刺激因子、趨化因子、或生長因子。在一些實施例中，一或多個單元之各者獨立地係腫瘤壞死因子、介白素、淋巴激素、干擾素、群落刺激因子、趨化因子、或生長因子。在一些實施例中，一或多個單元之各者獨立地包含與表10所述之胺基酸序列中之任一者具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%同一性之胺基酸序列。In some embodiments, the second binding portion is located at the C-terminus of the FAP antigen-binding portion. In some embodiments, the second binding portion is operatively linked to the C-terminus of the Fc fragment. In some embodiments, the second binding portion is linked to the Fc fragment via a linker. In some embodiments, the second binding portion binds to and/or activates a second target. In some embodiments, the second binding portion binds to and/or activates tumor-associated cell receptors. In some embodiments, the second binding portion comprises a first portion and a second portion, wherein each portion comprises one or more units. In some embodiments, one or more units comprise a first unit, a second unit, and/or a third unit. In some embodiments, each of the one or more units is identical. In some embodiments, one or more units are dissimilar. In some embodiments, the second binding portion is tumor necrosis factor, interleukin, lymphokine, interferon, community-stimulating factor, chemotherapeutic factor, or growth factor. In some embodiments, each of one or more units is independently tumor necrosis factor, interleukin, lymphokine, interferon, community-stimulating factor, chemotherapeutic factor, or growth factor. In some embodiments, each of one or more units independently comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any of the amino acid sequences described in Table 10.

在一些實施例中，第二結合部份包含第二抗原結合部份或細胞介素部份。在一些實施例中，第二結合部份包含細胞介素部份。在一些實施例中，細胞介素部份包含第一細胞介素部份及第二細胞介素部份。在一些實施例中，第一細胞介素部份包含第一細胞介素單元。在一些實施例中，第一細胞介素單元包含LIGHT單元或淋巴毒素-β單元。在一些實施例中，第二細胞介素部份包含第二細胞介素單元及第三細胞介素單元。在一些實施例中，第二細胞介素單元包含LIGHT單元、淋巴毒素-α單元、或淋巴毒素-β單元。在一些實施例中，第三細胞介素單元包含LIGHT單元、淋巴毒素-α單元、或淋巴毒素-β單元。在一些實施例中，第一細胞介素部份包含第一LIGHT單元，第二細胞介素單元包含第二LIGHT單元，且第三細胞介素單元包含第三LIGHT單元。在一些實施例中，第一LIGHT單元、第二LIGHT單元、及/或第三LIGHT單元各自獨立地包含SEQ ID NO: 17或18所述之胺基酸序列。在一些實施例中，第一細胞介素部份包含淋巴毒素-β單元，第二細胞介素單元包含淋巴毒素-α單元，且第三細胞介素單元包含淋巴毒素-β單元。在一些實施例中，淋巴毒素-β單元包含SEQ ID NO: 39所述之胺基酸序列，淋巴毒素-α單元包含SEQ ID NO: 40所述之胺基酸序列。在一些實施例中，雙特異性抗體或其抗原結合片段以不大於20 nM、15 nM、10 nM、或5 nM之解離常數(KD)結合至FAP。在一些實施例中，雙特異性抗體或其抗原結合片段以不大於20 nM、15 nM、10 nM、或5 nM之解離常數(KD)結合至LTßR。在一些實施例中，雙特異性抗體或其抗原結合片段幾乎不結合至人類或食蟹獼猴HVEM。在一些實施例中，雙特異性抗體或其抗原結合片段中之第二結合部份能夠降低與DcR3之結合親和力。在一些實施例中，雙特異性抗體或其抗原結合片段特異性結合至人類FAP，且/或不結合至DPPIV。在一些實施例中，第二結合部份結合及/或活化LTβR、HER2、PDL-1、PD-1、EGFR、VEGFR、VEGF、CCR8、OX-40、418B、血管生成素-2、IL-4Ra、BCMA、Blys、BTNO2、C5、CD122、CD13、CD133、CD137、CD138、CD16a、CD19、CD20、CD22、CD27、CD28、CD3、CD30、CD33、CD38、CD40、CD47、CD-8、CEA、CGPR/CGRPR、CSPGs、CTLA4、CTLA-4、DLL-4、EpCAM、因子IXa、因子X、GITR、GP130、Her3、HSG、ICOS、IGFl、IGFl/2、IGF-lR、IGF2、IGFR、IL-1、IL- 12、IL-12p40、IL-13、IL-l 7A、IL-1~、IL-23、IL-5、IL-6、IL-6R、Lag-3、LAG3、MAG、Met、NgR、NogoA、OMGp、OX40、PDGFR、PSMA、RGMA、RGMB、SARS-CoV-2、Te38、TIM-3、TNF、TNFa、TROP-2、TWEAK、或TRAIL。在一些實施例中，第二結合部份係第二抗原結合部份。在一些實施例中，第二抗原結合部份包含抗LTβR結合部份、抗HER2結合部份、抗PDL-1結合部份、抗PD-1結合部份、抗EGFR結合部份、抗VEGFR結合部份、抗VEGF結合部份、抗CCR8結合部份、抗OX-40結合部份、抗418B結合部份、抗血管生成素-2結合部份、抗IL-4Ra結合部份、抗BCMA結合部份、抗Blys結合部份、抗BTNO2結合部份、抗C5結合部份、抗CD122結合部份、抗CD13結合部份、抗CD133結合部份、抗CD137結合部份、抗CD138結合部份、抗CD16a結合部份、抗CD19結合部份、抗CD20結合部份、抗CD22結合部份、抗CD27結合部份、抗CD28結合部份、抗CD3結合部份、抗CD30結合部份、抗CD33結合部份、抗CD38結合部份、抗CD40結合部份、抗CD47結合部份、抗CD-8結合部份、抗CEA結合部份、抗CGPR/CGRPR結合部份、抗CSPGs結合部份、抗CTLA4結合部份、抗CTLA-4結合部份、抗DLL-4結合部份、抗EpCAM結合部份、抗因子IXa結合部份、抗因子X結合部份、抗GITR結合部份、抗GP130結合部份、抗Her3結合部份、抗HSG結合部份、抗ICOS結合部份、抗IGFl結合部份、抗IGFl/2結合部份、抗IGF-lR結合部份、抗IGF2結合部份、抗IGFR結合部份、抗IL-1結合部份、抗IL-12結合部份、抗IL-12p40結合部份、抗IL-13結合部份、抗IL-l 7A結合部份、抗IL-1~結合部份、抗IL-23結合部份、抗IL-5結合部份、抗IL-6結合部份、抗IL-6R結合部份、抗Lag-3結合部份、抗LAG3結合部份、抗MAG結合部份、抗Met結合部份、抗NgR結合部份、抗NogoA結合部份、抗OMGp結合部份、抗OX40結合部份、抗PDGFR結合部份、抗PSMA結合部份、抗RGMA結合部份、抗RGMB結合部份、抗SARS-CoV-2結合部份、抗Te38結合部份、抗TIM-3結合部份、抗TNF結合部份、抗TNFa結合部份、抗TROP-2結合部份、抗TWEAK結合部份、或抗TRAIL結合部份。In some embodiments, the second binding portion includes a second antigen-binding portion or an intercytokine portion. In some embodiments, the second binding portion includes an intercytokine portion. In some embodiments, the intercytokine portion includes a first intercytokine portion and a second intercytokine portion. In some embodiments, the first intercytokine portion includes a first intercytokine unit. In some embodiments, the first intercytokine unit includes a LIGHT unit or a lymphotoxin-β unit. In some embodiments, the second intercytokine portion includes a second intercytokine unit and a third intercytokine unit. In some embodiments, the second intercytokine unit includes a LIGHT unit, a lymphotoxin-α unit, or a lymphotoxin-β unit. In some embodiments, the third intercellular unit comprises a LIGHT unit, a lymphotoxin-α unit, or a lymphotoxin-β unit. In some embodiments, the first intercellular portion comprises a first LIGHT unit, the second intercellular unit comprises a second LIGHT unit, and the third intercellular unit comprises a third LIGHT unit. In some embodiments, the first LIGHT unit, the second LIGHT unit, and/or the third LIGHT unit each independently comprises the amino acid sequence described in SEQ ID NO: 17 or 18. In some embodiments, the first intercellular portion comprises a lymphotoxin-β unit, the second intercellular unit comprises a lymphotoxin-α unit, and the third intercellular unit comprises a lymphotoxin-β unit. In some embodiments, the lymphotoxin-β unit comprises the amino acid sequence described in SEQ ID NO: 39, and the lymphotoxin-α unit comprises the amino acid sequence described in SEQ ID NO: 40. In some embodiments, the bispecific antibody or its antigen-binding fragment binds to the FAP with a dissociation constant (KD) not greater than 20 nM, 15 nM, 10 nM, or 5 nM. In some embodiments, the bispecific antibody or its antigen-binding fragment binds to LTβR with a dissociation constant (KD) not greater than 20 nM, 15 nM, 10 nM, or 5 nM. In some embodiments, the bispecific antibody or its antigen-binding fragment binds almost no to human or cynomolgus monkey HVEM. In some embodiments, the second binding portion of a bispecific antibody or its antigen-binding fragment can reduce the binding affinity to DcR3. In some embodiments, the bispecific antibody or its antigen-binding fragment specifically binds to human FAP and/or does not bind to DPPIV. In some embodiments, the second binding portion binds to and/or activates LTβR, HER2, PDL-1, PD-1, EGFR, VEGFR, VEGF, CCR8, OX-40, 418B, angiopoietin-2, IL-4Ra, BCMA, Blys, BTNO2, C5, CD122, CD13, CD133, CD137, CD138, CD16a, CD19, CD20, CD22, CD27, CD28, CD3, CD30, CD33, CD38, CD40, CD47, CD-8, CEA, CGPR/CGRPR, CSPGs, CTLA4, CTLA-4, DLL-4, EpCAM, Factor IXa, Factor X, GITR, GP130, Her3, HSG, ICOS, IGF1, IGF1/2, IGF-1R, IGF2, IGFR, IL-1, IL- 12. IL-12p40, IL-13, IL-17A, IL-1~, IL-23, IL-5, IL-6, IL-6R, Lag-3, LAG3, MAG, Met, NgR, NogoA, OMGp, OX40, PDGFR, PSMA, RGMA, RGMB, SARS-CoV-2, Te38, TIM-3, TNF, TNFα, TROP-2, TWEAK, or TRAIL. In some embodiments, the second binding part is the second antigen binding part. In some embodiments, the second antigen-binding moiety includes an anti-LTβR binding moiety, an anti-HER2 binding moiety, an anti-PDL-1 binding moiety, an anti-PD-1 binding moiety, an anti-EGFR binding moiety, an anti-VEGFR binding moiety, an anti-VEGF binding moiety, an anti-CCR8 binding moiety, an anti-OX-40 binding moiety, an anti-418B binding moiety, an anti-angiogenic-2 binding moiety, an anti-IL-4Ra binding moiety, and an anti-BCMA binding moiety. Anti-Blys binding region, anti-BTNO2 binding region, anti-C5 binding region, anti-CD122 binding region, anti-CD13 binding region, anti-CD133 binding region, anti-CD137 binding region, anti-CD138 binding region, anti-CD16a binding region, anti-CD19 binding region, anti-CD20 binding region, anti-CD22 binding region, anti-CD27 binding region, anti-CD28 binding region, anti-CD3 binding region, anti-CD Anti-CD30 binding region, anti-CD33 binding region, anti-CD38 binding region, anti-CD40 binding region, anti-CD47 binding region, anti-CD-8 binding region, anti-CEA binding region, anti-CGPR/CGRPR binding region, anti-CSPGs binding region, anti-CTLA4 binding region, anti-CTLA-4 binding region, anti-DLL-4 binding region, anti-EpCAM binding region, anti-factor IXa binding region, anti-factor X binding region Parts, anti-GITR binding region, anti-GP130 binding region, anti-Her3 binding region, anti-HSG binding region, anti-ICOS binding region, anti-IGF1 binding region, anti-IGF1/2 binding region, anti-IGF-1R binding region, anti-IGF2 binding region, anti-IGFR binding region, anti-IL-1 binding region, anti-IL-12 binding region, anti-IL-12p40 binding region, anti-IL-13 binding region, anti-IL-1 7A binding region, anti-IL-1 binding region, anti-IL-23 binding region, anti-IL-5 binding region, anti-IL-6 binding region, anti-IL-6R binding region, anti-Lag-3 binding region, anti-LAG3 binding region, anti-MAG binding region, anti-Met binding region, anti-NgR binding region, anti-NogoA binding region, anti-OMGp binding region, anti-OX40 binding region, anti-PDGFR binding region, anti-PSMA binding region, anti-RGMA binding region, anti-RGMB binding region, anti-SARS-CoV-2 binding region, anti-Te38 binding region, anti-TIM-3 binding region, anti-TNF binding region, anti-TNFα binding region, anti-TROP-2 binding region, anti-TWEAK binding region, or anti-TRAIL binding region.

在一些實施例中，相對於比較物雙特異性抗體或其抗原結合片段，雙特異性抗體或其抗原結合片段展現出降低的與HVEM之結合親和力。在一些實施例中，相對於比較物雙特異性抗體或其抗原結合片段，雙特異性抗體或其抗原結合片段展現出降低的與DcR3之結合親和力。在一些實施例中，相對於比較物雙特異性抗體或其抗原結合片段，雙特異性抗體或其抗原結合片段展現出降低的與DPPIV之結合親和力。在一些實施例中，在使雙特異性抗體或其抗原結合片段與FAP表現性細胞接觸後，雙特異性抗體或其抗原結合片段誘導：次級淋巴器官(SLO)之形成、三級淋巴結構(TLS)之形成、免疫細胞之刺激、腫瘤細胞之細胞凋亡、癌症之治療、或其任何組合。In some embodiments, the bispecific antibody or its antigen-binding fragment exhibits reduced binding affinity to HVEM relative to the competitor bispecific antibody or its antigen-binding fragment. In some embodiments, the bispecific antibody or its antigen-binding fragment exhibits reduced binding affinity to DcR3 relative to the competitor bispecific antibody or its antigen-binding fragment. In some embodiments, the bispecific antibody or its antigen-binding fragment exhibits reduced binding affinity to DPPIV relative to the competitor bispecific antibody or its antigen-binding fragment. In some embodiments, after contacting a bispecific antibody or its antigen-binding fragment with FAP-expressing cells, the bispecific antibody or its antigen-binding fragment induces: the formation of secondary lymphoid organs (SLOs), the formation of tertiary lymphoid structures (TLSs), the stimulation of immune cells, apoptosis of tumor cells, cancer treatment, or any combination thereof.

本文亦提供一種融合蛋白，其中該融合蛋白係包含在本文所述之雙特異性抗體或其抗原結合片段中之任一者中。This article also provides a fusion protein, wherein the fusion protein is contained in either the bispecific antibody or its antigen-binding fragment described herein.

在一些實施例中，融合蛋白包含重鏈1 (HC1)區，其包含VH、重鏈恆定域1 (CH1)、及包含重鏈恆定域2 (CH2)及重鏈恆定域3 (CH3)之Fc片段。在一些實施例中，HC1區包含與如SEQ ID NO: 1、4、5、8、10、13、14、及15所示之胺基酸序列中之任一者包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列。在一些實施例中，HC1區包含與如SEQ ID NO: 5、8、10、13、14、及15所示之胺基酸序列中之任一者包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列。在一些實施例中，HC1區之Fc片段包含與Fc片段之C端融合的第二結合部份之一或多個單元。在一些實施例中，HC1區之Fc片段包含與HC1 Fc片段之C端融合的第二結合部份之第一單元。在一些實施例中，第二結合部份之第一單元係藉由第一連接子融合至Fc單元。在一些實施例中，第二結合部份係細胞介素部份，且其中細胞介素部份包含第一細胞介素單元。在一些實施例中，第一細胞介素單元包含LIGHT單元或淋巴毒素β單元。在一些實施例中，第一單元包含與表10所述之胺基酸序列中之任一者具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%同一性或與其同一之胺基酸序列。在一些實施例中，單元包含與SEQ ID NO: 17或SEQ ID NO: 39具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%同一性或與其同一之胺基酸序列。In some embodiments, the fusion protein includes a heavy chain 1 (HC1) region comprising VH, heavy chain constant 1 (CH1), and an Fc fragment comprising heavy chain constant 2 (CH2) and heavy chain constant 3 (CH3). In some embodiments, the HC1 region comprises an amino acid sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with or is identical to any of the amino acid sequences shown in SEQ ID NO: 1, 4, 5, 8, 10, 13, 14, and 15. In some embodiments, the HC1 region includes an amino acid sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with or identical to any of the amino acid sequences shown in SEQ ID NO: 5, 8, 10, 13, 14, and 15. In some embodiments, the Fc fragment of the HC1 region includes one or more units of a second binding portion fused to the C-terminus of the Fc fragment. In some embodiments, the Fc fragment of the HC1 region includes a first unit of a second binding portion fused to the C-terminus of the HC1 Fc fragment. In some embodiments, the first unit of the second binding portion is fused to the Fc unit via a first linker. In some embodiments, the second binding portion is an intercytokine portion, wherein the intercytokine portion includes a first intercytokine unit. In some embodiments, the first intercytokine unit comprises a LIGHT unit or a lymphotoxin β unit. In some embodiments, the first unit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with or identical to any of the amino acid sequences described in Table 10. In some embodiments, the unit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with or identical to SEQ ID NO: 17 or SEQ ID NO: 39.

在一些實施例中，融合蛋白包含重鏈2 (HC2)區，其包含VH、CH1、及包含CH1及CH3之Fc片段。在一些實施例中，HC2區包含與如SEQ ID NO: 1、4、6、9、11、12、13、及16所示之胺基酸序列中之任一者包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列。在一些實施例中，HC2區包含與如SEQ ID NO: 6、9、11、12、13、及16所示之胺基酸序列中之任一者包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列。在一些實施例中，HC2區之Fc片段包含與HC2 Fc片段之C端融合的第二結合部份之一或多個單元。在一些實施例中，HC2區之Fc片段包含第二結合部份之第二單元及第三單元。在一些實施例中，第二結合部份之第二單元係融合至HC2區之Fc片段。在一些實施例中，第二結合部份之第三單元係融合至第二結合部份之第二單元。在一些實施例中，第二結合部份之第二單元係藉由第二連接子融合至HC2區之Fc片段，且第三單元係藉由第三連接子融合至第二結合部份之第二單元。在一些實施例中，第二結合部份單元之第二單元及第三單元係串聯連接。在一些實施例中，第二結合部份係細胞介素部份，且其中細胞介素部份包含第二細胞介素單元及第三細胞介素單元。在一些實施例中，第二細胞介素單元包含LIGHT單元、淋巴毒素α單元、或淋巴毒素β單元。在一些實施例中，第三細胞介素單元包含LIGHT單元、淋巴毒素α單元、或淋巴毒素β單元。在一些實施例中，第二細胞介素單元包含LIGHT單元，且第三細胞介素單元包含LIGHT單元。在一些實施例中，第二細胞介素單元包含淋巴毒素α單元，且第三細胞介素單元包含淋巴毒素β單元。在一些實施例中，第二單元包含與表10所述之胺基酸序列中之任一者具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%同一性或與其同一之胺基酸序列。在一些實施例中，第二單元包含與SEQ ID NO: 17或SEQ ID NO: 40具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%同一性或與其同一之胺基酸序列。在一些實施例中，第三單元包含與表10所述之胺基酸序列中之任一者具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%同一性或與其同一之胺基酸序列。在一些實施例中，第三單元包含與SEQ ID NO: 17或SEQ ID NO: 39具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%同一性或與其同一之胺基酸序列。In some embodiments, the fusion protein includes a heavy chain 2 (HC2) region comprising VH, CH1, and an Fc fragment comprising CH1 and CH3. In some embodiments, the HC2 region comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identical or identical to any of the amino acid sequences shown in SEQ ID NO: 1, 4, 6, 9, 11, 12, 13, and 16. In some embodiments, the HC2 region comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identical or identical to any of the amino acid sequences shown in SEQ ID NO: 6, 9, 11, 12, 13, and 16. In some embodiments, the Fc segment of the HC2 region includes one or more units of a second bonding portion fused to the C-terminus of the HC2 Fc segment. In some embodiments, the Fc segment of the HC2 region includes a second unit and a third unit of the second bonding portion. In some embodiments, the second unit of the second bonding portion is fused to the Fc segment of the HC2 region. In some embodiments, the third unit of the second bonding portion is fused to the second unit of the second bonding portion. In some embodiments, the second unit of the second bonding portion is fused to the Fc segment of the HC2 region via a second connector, and the third unit is fused to the second unit of the second bonding portion via a third connector. In some embodiments, the second and third units of the second bonding portion unit are connected in series. In some embodiments, the second binding portion is an interleukin portion, wherein the interleukin portion includes a second interleukin unit and a third interleukin unit. In some embodiments, the second interleukin unit includes a LIGHT unit, a lymphotoxin alpha unit, or a lymphotoxin beta unit. In some embodiments, the third interleukin unit includes a LIGHT unit, a lymphotoxin alpha unit, or a lymphotoxin beta unit. In some embodiments, the second interleukin unit includes a LIGHT unit, and the third interleukin unit includes a LIGHT unit. In some embodiments, the second interleukin unit includes a lymphotoxin alpha unit, and the third interleukin unit includes a lymphotoxin beta unit. In some embodiments, the second unit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with or the same as any of the amino acid sequences described in Table 10. In some embodiments, the second unit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with or the same as any of the amino acid sequences described in Table 10. In some embodiments, the third unit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with or the same as any of the amino acid sequences described in Table 10. In some embodiments, the third unit comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to or the same as SEQ ID NO: 17 or SEQ ID NO: 39.

在一些實施例中，融合蛋白包含一或多個連接子。在一些實施例中，一或多個連接子之各者獨立地包含VH-CHl連接子ASTKGPSVFPLAPS；VL-CL連接子RTVAAPSVFIFPPS (SEQ ID NO: 91)；CH2-CH3連接子ISKAKGQPREPQ (SEQ ID NO: 92)；IgM尾部連接子KSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 93)；GGGGSGGGGSGGGGSGGGGT (SEQ ID NO: 94)；G；或(GGGGS)n (SEQ ID NO: 95)，其中n=l、2、3、4、5、6、7、8、9、或10。在一些實施例中，HC1區包含一或多個異二聚化修飾。在一些實施例中，一或多個異二聚化修飾係鈕(knob)修飾或孔(hole)修飾。在一些實施例中，一或多個異二聚化修飾係孔修飾。在一些實施例中，HC2區包含一或多個異二聚化修飾。在一些實施例中，一或多個異二聚化修飾係鈕(knob)修飾或孔(hole)修飾。在一些實施例中，一或多個異二聚化修飾係鈕修飾。In some embodiments, the fusion protein comprises one or more linkers. In some embodiments, each of the one or more linkers independently comprises the VH-CH1 linker ASTKGPSVFPLAPS; the VL-CL linker RTVAAPSVFIFPPS (SEQ ID NO: 91); the CH2-CH3 linker ISKAKGQPREPQ (SEQ ID NO: 92); the IgM tail linker KSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 93); GGGGSGGGGSGGGGSGGGGT (SEQ ID NO: 94); G; or (GGGGS)n (SEQ ID NO: 95), where n = 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the HC1 region comprises one or more heterodimerization modifications. In some embodiments, one or more isodimerization modifications are button or hole modifications. In some embodiments, one or more isodimerization modifications are hole modifications. In some embodiments, the HC2 region contains one or more isodimerization modifications. In some embodiments, one or more isodimerization modifications are button or hole modifications. In some embodiments, one or more isodimerization modifications are button modifications.

本文亦提供一種經單離多核苷酸，其包含編碼下列之一或多個核苷酸序列：本文所述之經單離抗FAP抗體或其FAP抗原結合片段中之任一者、本文所述之雙特異性抗體中之任一者、或本文所述之融合蛋白中之任一者。This document also provides a monosodium polynucleotide comprising encoding one or more of the following nucleotide sequences: any of the monosodium anti-FAP antibodies or their FAP antigen-binding fragments described herein, any of the bispecific antibodies described herein, or any of the fusion proteins described herein.

本文亦提供一種構築體，其包含本文所述之多核苷酸中之任一者。This article also provides a structure that includes any of the polynucleotides described herein.

本文亦提供一種抗體表現系統，其包含本文所述之構築體中之任一者或包含本文所述之多核苷酸中之任一者的載體。在一些實施例中，抗體表現系統係細胞表現系統。This document also provides an antibody expression system comprising any of the constructs described herein or a carrier comprising any of the polynucleotides described herein. In some embodiments, the antibody expression system is a cell expression system.

本文亦提供一種用於產生抗FAP抗體或其FAP抗原結合片段、雙特異性抗體或其抗原結合片段、或融合蛋白之方法，該方法包含：在適合表現該抗FAP抗體或其FAP抗原結合片段、雙特異性抗體或其抗原結合片段、或融合蛋白的條件下，使用本文所述之抗體表現系統中之任一者。This document also provides a method for generating an anti-FAP antibody or a FAP antigen-binding fragment thereof, a bispecific antibody or an antigen-binding fragment thereof, or a fusion protein, the method comprising: using any of the antibody expression systems described herein, under conditions suitable for expressing the anti-FAP antibody or a FAP antigen-binding fragment thereof, a bispecific antibody or an antigen-binding fragment thereof, or a fusion protein.

本文亦提供一種醫藥組成物，其中該醫藥組成物包含：本文所述之經單離抗FAP抗體或其FAP抗原結合片段中之任一者、本文所述之雙特異性抗體中之任一者、或本文所述之融合蛋白中之任一者；及醫藥上可接受之載劑。This article also provides a pharmaceutical composition comprising: any of the monoclonal anti-FAP antibodies or their FAP antigen-binding fragments described herein, any of the bispecific antibodies described herein, or any of the fusion proteins described herein; and a pharmaceutically acceptable delivery vehicle.

本文亦提供一種套組，其中該套組包含：本文所述之經單離抗FAP抗體或其FAP抗原結合片段中之任一者、本文所述之雙特異性抗體中之任一者、或本文所述之融合蛋白中之任一者，其包含本文所述之多核苷酸中之任一者；或本文所述之構築體中之任一者。This document also provides a kit comprising: any of the monoclonal FAP antibodies or their FAP antigen-binding fragments described herein, any of the bispecific antibodies described herein, or any of the fusion proteins described herein, which include any of the polynucleotides described herein; or any of the constructs described herein.

本文亦提供一種該抗FAP抗體或其FAP抗原結合片段、雙特異性抗體或其抗原結合片段、或融合蛋白於製造用於預防、診斷、或治療疾病、病症、或病況之治療劑的用途，其中：該抗FAP抗體或其FAP抗原結合片段係本文所述之經單離抗FAP抗體或其FAP抗原結合片段中之任一者，該雙特異性抗體或其抗原結合片段係本文所述之雙特異性抗體或其抗原結合片段中之任一者中之任一者，或該融合蛋白係本文所述之融合蛋白中之任一者。在一些實施例中，疾病、病症、或病況包含腫瘤疾病。在一些實施例中，至少一個腫瘤細胞表現FAP。在一些實施例中，腫瘤疾病係實體腫瘤。在一些實施例中，腫瘤疾病包含胃癌、肝癌、肺癌、結腸直腸癌、乳癌、前列腺癌、皮膚癌、骨癌、多發性骨髓瘤、神經膠質瘤、卵巢癌、胰臟癌、子宮頸癌、甲狀腺癌、喉癌、急性骨髓性白血病、慢性骨髓性白血病、急性淋巴母細胞白血病、慢性淋巴母細胞白血病、腦腫瘤、神經母細胞瘤、視網膜母細胞瘤、頭頸癌、唾液腺癌、及淋巴瘤。This document also provides the use of the anti-FAP antibody or its FAP antigen-binding fragment, a bispecific antibody or its antigen-binding fragment, or a fusion protein in the manufacture of a therapeutic agent for the prevention, diagnosis, or treatment of a disease, condition, or illness, wherein: the anti-FAP antibody or its FAP antigen-binding fragment is any of the monoclonal anti-FAP antibodies or their FAP antigen-binding fragments described herein; the bispecific antibody or its antigen-binding fragment is any of the bispecific antibodies or their antigen-binding fragments described herein; or the fusion protein is any of the fusion proteins described herein. In some embodiments, the disease, condition, or illness includes a neoplastic disease. In some embodiments, at least one tumor cell expresses FAP. In some embodiments, the neoplastic disease is a solid tumor. In some embodiments, oncological diseases include stomach cancer, liver cancer, lung cancer, colorectal cancer, breast cancer, prostate cancer, skin cancer, bone cancer, multiple myeloma, glioma, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, brain tumors, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and lymphoma.

本文亦提供一種用於治療有需要之對象的方法，其包含向該對象投予治療有效量的：本文所述之經單離抗FAP抗體或其FAP抗原結合片段中之任一者、本文所述之雙特異性抗體中之任一者、或本文所述之融合蛋白中之任一者、或本文所述之醫藥組成物中之任一者。This article also provides a method for treating a subject in need, comprising administering to the subject a therapeutically effective amount of: any of the monoclonal anti-FAP antibodies or their FAP antigen-binding fragments described herein, any of the bispecific antibodies described herein, any of the fusion proteins described herein, or any of the pharmaceutical compositions described herein.

本文亦提供一種用於降低腫瘤生長速率或腫瘤細胞數目之方法，其包含使腫瘤細胞與有效量的下列接觸：本文所述之經單離抗FAP抗體或其FAP抗原結合片段中之任一者、本文所述之雙特異性抗體中之任一者、或本文所述之融合蛋白中之任一者、或本文所述之醫藥組成物中之任一者。This article also provides a method for reducing tumor growth rate or tumor cell count, comprising contacting tumor cells with an effective amount of any of the following: any of the monoclonal anti-FAP antibodies or their FAP antigen-binding fragments described herein, any of the bispecific antibodies described herein, any of the fusion proteins described herein, or any of the pharmaceutical compositions described herein.

本文亦提供一種殺滅腫瘤細胞之方法，其包含使腫瘤細胞與有效量的下列接觸：本文所述之經單離抗FAP抗體或其FAP抗原結合片段中之任一者、本文所述之雙特異性抗體中之任一者、或本文所述之融合蛋白中之任一者、或本文所述之醫藥組成物中之任一者。This article also provides a method for killing tumor cells, comprising contacting the tumor cells with an effective amount of any of the following: any of the monoclonal anti-FAP antibodies or their FAP antigen-binding fragments described herein, any of the bispecific antibodies described herein, any of the fusion proteins described herein, or any of the pharmaceutical compositions described herein.

相關申請案之交互參照本申請案主張2023年12月1日申請之國際專利申請案第PCT/CN2023/135902號及2024年11月12日申請之國際專利申請案第PCT/CN2024/131641號之優先權權益，其等之各者之全部內容以引用方式併入本文中。 以引用方式併入序列表 Cross-referencing related applications: This application claims priority to International Patent Application No. PCT/CN2023/135902 filed on December 1, 2023, and International Patent Application No. PCT/CN2024/131641 filed on November 12, 2024, the entire contents of which are incorporated herein by reference. (Included in the sequence list by reference )

本申請案含有序列表，此序列表已經由Patent Center提交。標題為G24H06545W-PCT-SL-20241127.xml之序列表係建立於2024年11月27日，且檔案大小為112,357位元組，其全文特此以引用方式併入本文中。This application contains a sequence list, which has been submitted by the Patent Center. The sequence list, entitled G24H06545W-PCT-SL-20241127.xml, was created on November 27, 2024, and has a file size of 112,357 bytes. The full text of the file is hereby incorporated herein by reference.

以下更詳細地解釋本揭露。此實施方式並不意欲成為可實施本發明的所有不同方式或可添加至本發明的所有特徵之詳細目錄。例如，針對一個實施例所繪示的特徵可合併到其他實施例中，且針對特定實施例所繪示的特徵可從該實施例中刪除。另外，根據本揭露，對本文提出的各種實施例的許多變化及添加對於所屬技術領域中具有通常知識者而言將係顯而易見的，而不脫離本發明。因此，以下描述旨在說明本發明的一些特定實施例，而非窮舉地指定其所有排列、組合、及變化。The following provides a more detailed explanation of this disclosure. This embodiment is not intended to be a detailed catalogue of all different ways in which the invention can be implemented or all features that can be added to the invention. For example, a feature illustrated for one embodiment may be incorporated into other embodiments, and a feature illustrated for a particular embodiment may be deleted from that embodiment. Furthermore, according to this disclosure, many variations and additions to the various embodiments presented herein will be apparent to those skilled in the art without departing from the invention. Therefore, the following description is intended to illustrate certain specific embodiments of the invention, rather than to exhaustively specify all permutations, combinations, and variations thereof.

除非另有定義，否則本文中所使用之所有技術及科學用語具有與本揭露涉及之技術領域中具有通常知識者一般理解者相同的意義。儘管與本文所描述者相似或等效的任何方法及材料可用於測試本揭露的實踐中，但本文描述了較佳材料及方法。在描述及請求保護本揭露時，將使用以下用語。Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure relates. While any methods and materials similar to or equivalent to those described herein may be used in the practice of testing this disclosure, preferred materials and methods are described herein. The following terms will be used in describing and claiming protection for this disclosure.

如本文中所使用，用語「約(about)」當在數值之前時表示該值加上或減去20%、15%、10%、或5%的範圍。As used in this article, the term "about" before a numerical value indicates that the value is added to or subtracted from the range of 20%, 15%, 10%, or 5%.

用語「抗體(antibody)」（可與複數形式互換使用）係能夠透過位於免疫球蛋白分子之可變區中的至少一個抗原識別位點特異性結合至目標（諸如碳水化合物、多核苷酸、脂質、多肽等）之免疫球蛋白分子。如本文中所使用，用語「抗體」不僅包括完整（亦即全長）多株或單株抗體，且亦包括其抗原結合片段（例如Fab、Fab'、F(ab')2、Fv）、單鏈(scFv)、其突變體、包含抗體部份之融合蛋白、人源化抗體、嵌合抗體、雙鏈抗體(diabody)、奈米抗體(nanobody)、線性抗體、單鏈抗體、多特異性抗體（例如雙特異性抗體）、及包含具有所欲特異性之抗原識別位點的免疫球蛋白分子之任何其他經修飾構築體（包括抗體之醣基化變體、抗體之胺基酸序列變體、及經共價修飾之抗體）。抗體包括任何類別的抗體，諸如IgD、IgE、IgG、IgA、或IgM（或其子類），且抗體不需要屬於任何特定類別。取決於其重鏈恆定域的抗體胺基酸序列，免疫球蛋白可分為不同的類別。有五種主要類別的免疫球蛋白：IgA、IgD、IgE、IgG、及IgM，且此等中之數種可進一步分為子類（同型），諸如IgG1、IgG2、IgG3、IgG4、IgA1、及IgA2。將對應於不同類別的免疫球蛋白之重鏈恆定域分別稱為α、δ、ε、γ、及µ。不同類別的免疫球蛋白之次單元結構及三維構形係熟知的。The term "antibody" (which can be used interchangeably with the plural form) refers to an immunoglobulin molecule that can specifically bind to a target (such as carbohydrates, polynucleotides, lipids, polypeptides, etc.) through at least one antigen recognition site located in the variable region of an immunoglobulin molecule. As used herein, the term "antibody" includes not only full-length (i.e., full-length) multi- or single-celled antibodies, but also their antigen-binding fragments (e.g., Fab, Fab', F(ab')2, Fv), single-stranded (scFv), their mutants, fusion proteins containing antibody portions, humanized antibodies, chimeric antibodies, diabody antibodies, nanobody antibodies, linear antibodies, single-stranded antibodies, multispecific antibodies (e.g., bispecific antibodies), and any other modified constructs of an immunoglobulin molecule containing an antigen recognition site with desired specificity (including glycosylated variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies). Antibodies include any class of antibodies, such as IgD, IgE, IgG, IgA, or IgM (or their subclasses), and antibodies do not need to belong to any particular class. Immunoglobulins can be classified into different classes based on the amino acid sequence of their heavy chain constants. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constants corresponding to different classes of immunoglobulins are respectively called α, δ, ε, γ, and µ. The subunit structures and three-dimensional conformations of different classes of immunoglobulins are well known.

在本揭露中，「胺基酸」係指含有胺(-NH 2)及羧基(-COOH)官能基連同對各胺基酸具有特異性的側鏈的有機化合物。胺基酸的名稱以標準的單字母或三字母代碼表示，如下所示： 胺基酸名稱 三字母代碼 單字母代碼 丙胺酸 Ala A 半胱胺酸 Cys C 天冬胺酸 Asp D 麩胺酸 Glu E 苯丙胺酸 Phe F 甘胺酸 Gly G 組胺酸 His H 異白胺酸 Ile I 離胺酸 Lys K 白胺酸 Leu L 甲硫胺酸 Met M 天冬醯胺酸 Asn N 脯胺酸 Pro P 麩醯胺酸 Gln Q 精胺酸 Arg R 絲胺酸 Ser S 蘇胺酸 Thr T 纈胺酸 Val V 色胺酸 Trp W 酪胺酸 Tyr Y In this disclosure, "amino acid" refers to an organic compound containing amine ( -NH₂ ) and carboxyl (-COOH) functional groups along with side chains specific to each amino acid. The names of amino acids are represented by standard single-letter or three-letter codes, as shown below: Amino acid name Three-letter code Single letter code alanine Ala A Cysteine Cys C Aspartic acid Asp D glutamic acid Glu E Phenylan Phe F Glycine Gly G histidine His H Isoleucine Ile I lysine Lys K Leucine Leu L Methionine Met M aspartic acid Asn N proline Pro P glutamic acid Gln Q Arginine Arg R Serine Ser S threonine Thr T citric acid Val V tryptophan Trp W Tyrosine Tyr Y

如本文中所使用，用語「雙特異性抗體(bispecific antibody)」或「雙特異性分子(bispecific molecule)」係指對兩個特定表位表現出雙重結合特異性及親和力的抗體或分子、或其中所有抗體對兩個特定表位表現出雙重結合特異性及親和力的抗體的組成物。As used herein, the terms "bispecific antibody" or "bispecific molecule" refer to an antibody or molecule that exhibits dual binding specificity and affinity for two specific epitopes, or a composition of antibodies in which all antibodies exhibit dual binding specificity and affinity for two specific epitopes.

如本文中所使用，用語「比較物結合劑(comparator binding agent)」係指可用以在本文所述之活性及/或檢定中提供參考水平的適當的相應對照或競爭性結合劑。例如，本文所述之抗FAP抗體之比較物結合劑可係結合至FAP之不同抗體。As used herein, the term "comparator binding agent" refers to an appropriate corresponding or competitive binding agent that can be used to provide a reference level in the activities and/or assays described herein. For example, the comparator binding agent for the anti-FAP antibody described herein could be a different antibody bound to FAP.

「Fc區(Fc region)」（可結晶片段區）或「Fc域(Fc domain)」或「Fc片段(Fc fragment)」係指抗體之重鏈的C端區，其介導免疫球蛋白與宿主組織或因子的結合，包括與位於免疫系統之各種細胞（例如效應細胞）上的Fc受體或與經典補體系統的第一組分(Clq)的結合。因此，Fc區包含排除第一恆定區免疫球蛋白域（例如CH1或CL）之抗體恆定區。在IgG、IgA、及IgD抗體同型中，Fc區包含兩個同一之蛋白質片段，該等蛋白質片段衍生自抗體之兩條重鏈的第二(CH2)恆定域及第三(CH3)恆定域；IgM及IgE Fc區在各多肽鏈中包含三個重鏈恆定域（CH域2至4）。對於IgG，Fc區包含免疫球蛋白域Cγ2及Cγ3及介於Cγ1與Cγ2之間的鉸鏈。儘管免疫球蛋白重鏈Fc區的邊界可能有所不同，但人類IgG重鏈Fc區通常定義為從位置C226或P230的胺基酸殘基（或介於該兩個胺基酸之間的胺基酸）延伸到重鏈的羧基端，其中編號係根據Kabat中的EU索引。人類IgG Fc區的CH2域從約胺基酸231延伸至約胺基酸340，而CH3域定位於Fc區中之CH2域的C端側，亦即，其從IgG的約胺基酸341延伸至約胺基酸447。如本文中所使用，Fc區可係天然序列Fc（包括任何同種異型變體）或變體Fc（例如非天然存在的Fc）。Fc亦可指單離的或在包含Fc之蛋白質多肽的情況下的此區。The "Fc region" (crystallizable fragment region), "Fc domain," or "Fc fragment" refers to the C-terminal region of the antibody heavy chain, which mediates the binding of immunoglobulins to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells) or to the first component (Clq) of the classical complement system. Therefore, the Fc region contains an antibody-stationary region excluding the first-stationary immunoglobulin domain (e.g., CH1 or CL). In IgG, IgA, and IgD antibody isotypes, the Fc region contains two identical protein fragments derived from the second (CH2) and third (CH3) constant domains of the two heavy chains of the antibody. The IgM and IgE Fc regions contain three heavy chain constant domains (CH domains 2 to 4) in each polypeptide chain. For IgG, the Fc region contains immunoglobulin domains Cγ2 and Cγ3 and a hinge between Cγ1 and Cγ2. Although the boundaries of the immunoglobulin heavy chain Fc region may vary, the human IgG heavy chain Fc region is generally defined as extending from the amino acid residue at position C226 or P230 (or an amino acid between these two amino acids) to the carboxyl terminus of the heavy chain, where the designation is based on the EU index in Kabat. The CH2 domain of the human IgG Fc region extends from amino acid 231 to amino acid 340, while the CH3 domain is located on the C-terminal side of the CH2 domain in the Fc region, that is, it extends from amino acid 341 to amino acid 447 of IgG. As used herein, the Fc region can be a native sequence Fc (including any allotype variant) or a variant Fc (e.g., a non-naturally occurring Fc). Fc can also refer to this region either singly or in the case of a protein-peptide containing Fc.

當關於肽、多肽、或蛋白質的胺基酸序列使用時，用語「融合(fusion)」係指藉由例如化學鍵結或重組手段將來自二或更多個不同來源的二或更多個胺基酸序列組合成單一胺基酸序列。融合胺基酸序列可由兩個編碼多核苷酸序列的基因重組來產生，且可藉由將含有重組多核苷酸的構築體引入宿主細胞中的方法來表現。因此，如本文中所使用，「融合蛋白(fusion protein)」可指包含來自至少兩種不同蛋白質之蛋白質域的雜合多肽。雖然「融合蛋白」及「免疫細胞介素(immunocytokine)」在本文中可互換使用，但應理解，並非本文所提供之所有融合蛋白均係免疫細胞介素。在一些情況下，融合蛋白亦可指二或更多個肽、多肽、或蛋白質的錯合物（例如結合劑），其中該等肽、多肽、或蛋白質中之至少一者具有將來自二或更多個不同來源的二或更多個胺基酸序列融合成單一胺基酸序列的胺基酸序列。When referring to the amino acid sequences of peptides, polypeptides, or proteins, the term "fusion" means the combination of two or more amino acid sequences from two or more different sources into a single amino acid sequence by means of, for example, chemical bonding or recombination. Fusion amino acid sequences can be generated by recombination of two genes encoding polynucleotide sequences and can be expressed by introducing a construct containing the recombinated polynucleotide into a host cell. Therefore, as used herein, "fusion protein" can refer to a hybrid polypeptide containing protein domains from at least two different proteins. Although "fusion protein" and "immunocytokine" are used interchangeably herein, it should be understood that not all fusion proteins presented herein are immunocytokines. In some cases, a fusion protein may also refer to a complex (e.g., a binder) of two or more peptides, polypeptides, or proteins, wherein at least one of such peptides, polypeptides, or proteins has an amino acid sequence that fuses two or more amino acid sequences from two or more different sources into a single amino acid sequence.

用語「醫藥上可接受之載劑(pharmaceutically acceptable carrier)」係指醫藥組成物中除活性成分之外的對對象無毒的成分。The term "pharmaceutically acceptable carrier" refers to a pharmaceutical composition in which the components other than the active ingredient are non-toxic to the target organism.

用語「治療/預防(treating/preventing)」（及其語法變體）係指試圖改變經治療個體中疾病的自然進展，且可係在臨床病理過程期間實施的預防或臨床干預。治療的所欲效果包括但不限於預防疾病的發生或再發、減輕症狀、緩解疾病的任何直接或間接的病理後果、預防轉移、減緩疾病進展的速度、改善或減輕疾病狀態、及緩解或改善預後。在一些實施例中，本揭露的抗體可用於延緩疾病的發展或延緩病症的進展。The term "treating/preventing" (and its grammatical variations) refers to an attempt to alter the natural progression of a disease in a treated individual, and may be prevention or clinical intervention implemented during the clinical pathological process. The desired effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, mitigating any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving prognosis. In some embodiments, the antibodies disclosed herein may be used to delay the development of disease or slow the progression of symptoms.

序列同一性百分比(%)係定義為候選序列中分別與參考多肽序列或多核苷酸序列中之胺基酸殘基或核酸同一的胺基酸殘基或核酸之百分比，其係在比對序列並引入間隙（若需要）之後以達成最大序列同一性百分比，且當涉及多肽序列時不考慮將任何保守性取代作為序列同一性之一部分。出於判定胺基酸序列同一性百分比之目的的比對可使用公開可得的電腦軟體以各種方式達成，諸如BLAST、BLAST-2、ALIGN、或Megalign (DNASTAR)軟體。例如，可使用NCBI資料庫的BLAST程式來判定同一性。Sequence identity percentage (%) is defined as the percentage of amino acid residues or nucleic acids in a candidate sequence that are identical to those in a reference polypeptide or polynucleotide sequence. It is achieved by aligning sequences and introducing gaps (if necessary) to reach the maximum sequence identity percentage, and when polypeptide sequences are involved, no conservation is considered as part of the sequence identity. Alignments for determining the percentage of amino acid sequence identity can be performed in various ways using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR). For example, the BLAST program from the NCBI database can be used to determine identity.

本揭露的其他特徵及優點將從以下詳細描述及圖式、及從申請專利範圍中顯而易見。 FAP 結合劑 Other features and advantages disclosed herein will be apparent from the following detailed description and figures, and from the scope of the patent application. FAP binder

本揭露提供纖維母細胞活化蛋白(fibroblast activation protein, FAP)結合劑（例如抗FAP抗體或其抗原結合片段、其雙特異性抗體、或其融合蛋白），該等結合劑結合至FAP，且在一些實施例中，特異性結合至FAP中所包含的FAP多肽、FAP多肽片段、FAP肽、或FAP表位。在一些實施例中，諸如人類FAP結合劑的FAP結合劑（例如抗FAP抗體或其抗原結合片段、雙特異性抗體、或其融合蛋白）可結合至哺乳動物（例如人類）細胞（包括FAP表現性細胞，諸如FAP表現性腫瘤細胞）的表面上表現的FAP。在一些實施例中，本文描述結合至FAP（諸如人類FAP或其部分）之FAP結合劑（例如抗FAP抗體或其抗原結合片段、雙特異性抗體、或其融合蛋白）。在一些實施例中，FAP係人類FAP。在一些實施例中，FAP結合劑係人類FAP結合劑（例如抗FAP抗體或其抗原結合片段、雙特異性抗體、或其融合蛋白）。This disclosure provides fibroblast activation protein (FAP) binding agents (e.g., anti-FAP antibodies or antigen-binding fragments thereof, bispecific antibodies thereof, or fusion proteins thereof) that bind to FAP, and in some embodiments, specifically bind to FAP polypeptides, FAP polypeptide fragments, FAP peptides, or FAP epitopes contained in the FAP. In some embodiments, FAP binding agents such as human FAP binding agents (e.g., anti-FAP antibodies or antigen-binding fragments thereof, bispecific antibodies thereof, or fusion proteins thereof) can bind to FAP expressed on the surface of mammalian (e.g., human) cells (including FAP-expressing cells, such as FAP-expressing tumor cells). In some embodiments, this document describes FAP binders (e.g., anti-FAP antibodies or antigen-binding fragments thereof, bispecific antibodies, or fusion proteins thereof) that bind to FAPs (such as human FAPs or portions thereof). In some embodiments, the FAP is a human FAP. In some embodiments, the FAP binder is a human FAP binder (e.g., anti-FAP antibodies or antigen-binding fragments thereof, bispecific antibodies, or fusion proteins thereof).

在一些實施例中，FAP結合劑（例如抗FAP抗體或其抗原結合片段、雙特異性抗體、或其融合蛋白）結合FAP表現性細胞之FAP表位（例如目標FAP表位）。在一些實施例中，FAP結合劑（例如抗FAP抗體或其抗原結合片段、雙特異性抗體、或其融合蛋白）選擇性地結合FAP表現性細胞之FAP表位，而非競爭性分子中之任一者或組合之表位，諸如促腫瘤細胞表面分子（例如DPPIV）。In some embodiments, FAP binders (e.g., anti-FAP antibodies or their antigen-binding fragments, bispecific antibodies, or their fusion proteins) bind to FAP epitopes (e.g., target FAP epitopes) on FAP-expressing cells. In some embodiments, FAP binders (e.g., anti-FAP antibodies or their antigen-binding fragments, bispecific antibodies, or their fusion proteins) selectively bind to FAP epitopes on FAP-expressing cells, rather than epitopes of any or a combination of competing molecules, such as tumor-promoting cell surface molecules (e.g., DPPIV).

如本文所述之FAP可係具有如 SEQ ID NO: 43（UniProt識別符P51685）所述之胺基酸序列的人類FAP。然而，FAP來自對應於本文所述之組成物及方法中之任何對象之物種的任何物種。例如，如本文所述之FAP可係具有如 SEQ ID NO: 44(NCBI XP_005573377.1)所述之胺基酸序列的食蟹獼猴FAP（恆河獼猴(Macaca mulatta)）；或具有如 SEQ ID NO: 45（UniProt識別符Uniprot P97321）所述之胺基酸序列的小鼠FAP（家鼷鼠(Mus musculus)）。 The FAP described herein may be a human FAP having the amino acid sequence described in SEQ ID NO: 43 (UniProt identifier P51685). However, the FAP may be from any species of any species corresponding to any object of the composition and method described herein. For example, the FAP described herein may be a cynomolgus monkey FAP (Macaca mulatta) having the amino acid sequence described in SEQ ID NO: 44 (NCBI XP_005573377.1); or a mouse FAP (Mus musculus) having the amino acid sequence described in SEQ ID NO: 45 (UniProt identifier Uniprot P97321).

具體地，本揭露涵蓋的FAP已顯示在癌症相關基質細胞的細胞表面及次級淋巴器官中的纖維母細胞網狀細胞上高度表現，但在正常組織中具有非常有限的表現。因此，靶向FAP特異性靶向腫瘤微環境中的腫瘤基質細胞，諸如腫瘤內皮及癌相關纖維母細胞。Specifically, the FAP disclosed herein has been shown to exhibit high expression on the cell surface of cancer-associated stromal cells and fibroblasts in secondary lymphoid organs, but has very limited expression in normal tissues. Therefore, targeting FAP specifically targets tumor stromal cells in the tumor microenvironment, such as tumor endothelial cells and cancer-associated fibroblasts.

因此，本揭露涵蓋的FAP相關疾病、病症、或病況係指特徵在於FAP的異常、上調、或選擇性表現的任何疾病、病症、或病況，及/或替代地所欲的係減少FAP及/或FAP表現性細胞之表現的任何疾病、病症、或病況。本文進一步描述例示性的FAP相關疾病、病症、或病況。Therefore, the FAP-related diseases, conditions, or illnesses covered by this disclosure refer to any disease, condition, or illness characterized by abnormal, upregulated, or selective expression of FAP, and/or alternatively, any disease, condition, or illness intended to reduce FAP and/or the expression of FAP-expressing cells. This document further describes illustrative FAP-related diseases, conditions, or illnesses.

在一些實施例中，本文所提供之FAP結合劑結合或特異性結合至FAP中所包含的目標FAP多肽、FAP多肽片段、FAP肽、或FAP表位。在一些實施例中，本文所提供之FAP結合劑特異性結合至目標FAP表位。目標表位可係連續的或非連續的，且可藉由所屬技術領域中具有通常知識者已知的方法來判定，該方法包括結合至肽的抗體的流式細胞術、氫-氘交換、丙胺酸掃描、及/或X射線晶體學。目標表位可包含胺基酸殘基或由胺基酸殘基組成，該等胺基酸殘基藉由本文所述之結合至FAP的FAP結合劑來判定。目標表位可係包含胺基酸殘基或由胺基酸殘基所組成之表位，胺基酸殘基藉由表位分倉(epitope binning)判定。目標表位可係包含胺基酸殘基或由胺基酸殘基組成的表位，該等胺基酸殘基藉由結合至FAP肽-奈米抗體錯合物的FAP結合劑來判定。目標表位可係包含胺基酸殘基或由胺基酸殘基組成的表位，該等胺基酸殘基藉由噬菌體展示對結合至FAP的FAP結合劑進行篩選來判定。目標表位可係包含胺基酸殘基或由胺基酸殘基組成的表位，該等胺基酸殘基藉由電腦學習模型對結合至FAP的FAP結合劑進行電腦模擬篩選來判定。目標表位可係包含胺基酸殘基或由胺基酸殘基組成的表位，該等胺基酸殘基藉由在競爭性檢定或非競爭性檢定中的結合至FAP的FAP結合劑來判定。In some embodiments, the FAP binders provided herein bind to or specifically bind to target FAP peptides, FAP peptide fragments, FAP peptides, or FAP epitopes contained in an FAP. In some embodiments, the FAP binders provided herein specifically bind to target FAP epitopes. Target epitopes may be continuous or discontinuous and can be determined by methods known to those skilled in the art, including flow cytometry, hydrogen-deuterium exchange, alanine scanning, and/or X-ray crystallography of antibodies bound to peptides. Target epitopes may contain or consist of amino acid residues, which are determined by the FAP binders described herein that bind to the FAP. Target epitopes may be epitopes containing or composed of amino acid residues, which are determined by epitope binning. Target epitopes may be epitopes containing or composed of amino acid residues, which are determined by FAP binding agents to FAP peptide-nanoantibody complexes. Target epitopes may be epitopes containing or composed of amino acid residues, which are determined by phage display screening of FAP binding agents to FAP. The target epitope may be an epitope containing or composed of amino acid residues, which are determined by computer-simulated screening of FAP binders bound to FAP using a computer learning model. The target epitope may be an epitope containing or composed of amino acid residues, which are determined by FAP binders bound to FAP in competitive or non-competitive testing.

在一些實施例中，目標表位包含與 表 5所述之序列中之任一者約60%、約65%、約70%、約75%、約80%、約85%、約90%、約95%、約97%、約98%、約99%同一或與其同一之胺基酸序列。在一些實施例中，目標表位包含1個胺基酸、2個胺基酸、3個胺基酸、4個胺基酸、5個胺基酸、6個胺基酸、7個胺基酸、8個胺基酸、9個胺基酸、10個胺基酸、11個胺基酸、12個胺基酸、13個胺基酸、14個胺基酸、15個胺基酸、16個胺基酸、17個胺基酸、18個胺基酸、19個胺基酸、或20個胺基酸的胺基酸序列，此等胺基酸係連續的或非連續的且選自 表 5所述之序列中之任一者。 In some embodiments, the target epitope contains an amino acid sequence that is about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, or about 99% the same as any of the sequences described in Table 5. In some embodiments, the target epitope comprises an amino acid sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids, which may be continuous or discontinuous and are selected from any of the sequences described in Table 5 .

在一些實施例中，本文所提供之FAP結合劑可對FAP具有強結合親和力及/或特異性。在一些實施例中，本文所提供之FAP結合劑可對FAP具有比其他競爭性分子（諸如二肽基肽酶4 (DPPIV)）強的結合親和力及/或特異性。在一些實施例中，本文所提供之FAP結合劑可對FAP表現性細胞具有強結合親和力及/或特異性。在一些實施例中，FAP在腫瘤細胞上表現，且/或FAP表現性細胞係FAP+基質細胞。In some embodiments, the FAP binders provided herein may have a strong binding affinity and/or specificity for FAP. In some embodiments, the FAP binders provided herein may have a stronger binding affinity and/or specificity for FAP than other competing molecules (such as dipeptidyl peptidase 4 (DPPIV)). In some embodiments, the FAP binders provided herein may have a strong binding affinity and/or specificity for FAP-expressing cells. In some embodiments, FAP is expressed on tumor cells, and/or the FAP-expressing cells are FAP+ stromal cells.

FAP結合劑包含抗FAP抗體、FAP抗原結合片段或其FAP抗原結合部份、包含抗FAP抗體或FAP抗原結合部份之雙特異性抗體、及/或其融合蛋白。在一些實施例中，本文所提供之FAP結合劑能夠結合二或更多個目標。在一些實施例中，本文所提供之FAP結合劑能夠結合FAP抗原且結合至少一個額外目標。在一些實施例中，本文所提供之FAP結合劑能夠結合FAP抗原且交聯及/或活化在腫瘤相關細胞上表現的額外目標。在一些實施例中，FAP結合劑與腫瘤相關細胞上表現的第二目標的結合交聯及/或活化額外目標，藉以實現抗腫瘤活性。本文進一步詳細描述FAP結合劑及其活性的實例。FAP conjugates comprise anti-FAP antibodies, FAP antigen-binding fragments or their FAP antigen-binding portions, bispecific antibodies comprising anti-FAP antibodies or FAP antigen-binding portions, and/or their fusion proteins. In some embodiments, the FAP conjugates provided herein are capable of binding two or more targets. In some embodiments, the FAP conjugates provided herein are capable of binding FAP antigens and binding at least one additional target. In some embodiments, the FAP conjugates provided herein are capable of binding FAP antigens and cross-linking and/or activating additional targets expressed on tumor-associated cells. In some embodiments, the FAP conjugate cross-links with and/or activates additional targets expressed on tumor-associated cells to achieve antitumor activity. This article provides further details on FAP binders and examples of their activity.

在一些實施例中，本文所述之FAP結合劑（例如抗FAP抗體或其抗原結合片段、雙特異性抗體、或其融合蛋白）與包含本文所述之抗體中之任一者的VH區、VL區、VH CDR1、VH CDR2、VH CDR3、VL CDR1、VL CDR2、及/或VL CDR3（諸如 表 6 至表8中描繪的VH區、VL區、VH CDR1、VH CDR2、VH CDR3、VL CDR1、VL CDR2、及/或VL CDR3的胺基酸序列）的FAP結合劑（例如抗FAP抗體或其抗原結合片段、雙特異性抗體、或其融合蛋白）競爭與FAP（諸如人類FAP）的結合。因此，在一些實施例中，本文所述之FAP結合劑（例如抗FAP抗體或其抗原結合片段、雙特異性抗體、或其融合蛋白）與包含來自下列的一個、兩個、及/或三個VH CDR及/或一個、兩個、及/或三個VL CDR的FAP結合劑（例如抗FAP抗體或其抗原結合片段、雙特異性抗體、或其融合蛋白）競爭與FAP（諸如人類FAP）的結合：(a)指定為9E3嵌合體(ABC1139)的抗體；(b)指定為ABC1931的抗體；(c)指定為ABC1930的抗體；(d)指定為ABC1773的抗體；(e)指定為ABC1947的抗體；(f)指定為ABC2066的抗體；(g)指定為ABC2067的抗體；或(h)指定為ABC2097的抗體，如 表 6 或表7中所示。在一些實施例中，本文所述之FAP結合劑（例如抗FAP抗體或其抗原結合片段、雙特異性抗體、或其融合蛋白）與包含來自下列的一個、兩個、及/或三個VH CDR及一個、兩個、及/或三個VL CDR的FAP結合劑（例如抗FAP抗體或其抗原結合片段、雙特異性抗體、或其融合蛋白）競爭與FAP（諸如人類FAP）的結合：(a)指定為9E3嵌合體(ABC1139)的抗體；(b)指定為ABC1931的抗體；(c)指定為ABC1930的抗體；(d)指定為ABC1773的抗體；(e)指定為ABC1947的抗體；(f)指定為ABC2066的抗體；(g)指定為ABC2067的抗體；或(h)指定為ABC2097的抗體，如 表 6 或表7中所示。在一些實施例中，本文所述之FAP結合劑（例如抗FAP抗體或其抗原結合片段、雙特異性抗體、或其融合蛋白）與包含來自下列的VH區及VL區的FAP結合劑（例如抗FAP抗體或其抗原結合片段、雙特異性抗體、或其融合蛋白）競爭與FAP（諸如人類FAP）的結合：(a)指定為9E3嵌合體(ABC1139)的抗體；(b)指定為ABC1931的抗體；(c)指定為ABC1930的抗體；(d)指定為ABC1773的抗體；(e)指定為ABC1947的抗體；(f)指定為ABC2066的抗體；(g)指定為ABC2067的抗體；或(h)指定為ABC2097的抗體，如 表 8中所示。 In some embodiments, the FAP binders described herein (e.g., anti-FAP antibodies or their antigen-binding fragments, bispecific antibodies, or their fusion proteins) compete with FAP binders (e.g., anti-FAP antibodies or their antigen-binding fragments, bispecific antibodies, or their fusion proteins ) that contain the VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 of any of the antibodies described herein (such as the amino acid sequences of the VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 as depicted in Tables 6 to 8) for binding to FAP (such as human FAP). Therefore, in some embodiments, the FAP conjugates described herein (e.g., anti-FAP antibodies or their antigen-binding fragments, bispecific antibodies, or their fusion proteins) contain one, two, and/or three VH CDRs and/or one, two, and/or three VLs derived from the following. CDR's FAP binders (e.g., anti-FAP antibodies or their antigen-binding fragments, bispecific antibodies, or their fusion proteins) compete with binding to FAP (such as human FAP) in the following ways: (a) an antibody designated as the 9E3 chimeric (ABC1139); (b) an antibody designated as ABC1931; (c) an antibody designated as ABC1930; (d) an antibody designated as ABC1773; (e) an antibody designated as ABC1947; (f) an antibody designated as ABC2066; (g) an antibody designated as ABC2067; or (h) an antibody designated as ABC2097, as shown in Table 6 or Table 7 . In some embodiments, the FAP conjugate described herein (e.g., an anti-FAP antibody or its antigen-binding fragment, a bispecific antibody, or its fusion protein) comprises one, two, and/or three VH CDRs and one, two, and/or three VLs derived from the following. CDR's FAP binders (e.g., anti-FAP antibodies or their antigen-binding fragments, bispecific antibodies, or their fusion proteins) compete with binding to FAP (such as human FAP) in the following ways: (a) an antibody designated as the 9E3 chimeric (ABC1139); (b) an antibody designated as ABC1931; (c) an antibody designated as ABC1930; (d) an antibody designated as ABC1773; (e) an antibody designated as ABC1947; (f) an antibody designated as ABC2066; (g) an antibody designated as ABC2067; or (h) an antibody designated as ABC2097, as shown in Table 6 or Table 7 . In some embodiments, the FAP binders described herein (e.g., anti-FAP antibodies or their antigen-binding fragments, bispecific antibodies, or their fusion proteins) compete with FAP binders (e.g., anti-FAP antibodies or their antigen-binding fragments, bispecific antibodies, or their fusion proteins) comprising VH and VL regions from the following: (a) an antibody designated as 9E3 chimera (ABC1139); (b) an antibody designated as ABC1931; (c) an antibody designated as ABC1930; (d) an antibody designated as ABC1773; (e) an antibody designated as ABC1947; (f) an antibody designated as ABC2066; (g) an antibody designated as ABC2067; or (h) an antibody designated as ABC2097, as shown in Table 8 .

在一些實施例中，本文所述之FAP結合劑（例如抗FAP抗體或其抗原結合片段、雙特異性抗體、或其融合蛋白）包含本文所述之抗體中之任一者的VH區、VL區、VH CDR1、VH CDR2、VH CDR3、VL CDR1、VL CDR2、及/或VL CDR3（諸如 表 6 至表8中描繪的VH區、VL區、VH CDR1、VH CDR2、VH CDR3、VL CDR1、VL CDR2、及/或VL CDR3的胺基酸序列）。因此，在一些實施例中，本文所述之FAP結合劑（例如抗FAP抗體或其抗原結合片段、雙特異性抗體、或其融合蛋白）包含來自下列的一個、兩個、及/或三個重鏈CDR及/或一個、兩個、及/或三個輕鏈CDR：(a)指定為9E3嵌合體(ABC1139)的抗體；(b)指定為ABC1931的抗體；(c)指定為ABC1930的抗體；(d)指定為ABC1773的抗體；(e)指定為ABC1947的抗體；(f)指定為ABC2066的抗體；(g)指定為ABC2067的抗體；或(h)指定為ABC2097的抗體，如 表 6 至表7中所示。在一些實施例中，本文所述之FAP結合劑（例如抗FAP抗體或其抗原結合片段、雙特異性抗體、或其融合蛋白）包含來自下列的一個、兩個、及/或三個重鏈CDR及一個、兩個、及/或三個輕鏈CDR：(a)指定為9E3嵌合體(ABC1139)的抗體；(b)指定為ABC1931的抗體；(c)指定為ABC1930的抗體；(d)指定為ABC1773的抗體；(e)指定為ABC1947的抗體；(f)指定為ABC2066的抗體；(g)指定為ABC2067的抗體；或(h)指定為ABC2097的抗體，如 表 6 至表7中所示。 In some embodiments, the FAP conjugates described herein (e.g., anti-FAP antibodies or their antigen-binding fragments, bispecific antibodies, or their fusion proteins) include the VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 of any of the antibodies described herein (such as the amino acid sequences of the VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 as depicted in Tables 6 to 8). Therefore, in some embodiments, the FAP conjugates described herein (e.g., anti-FAP antibodies or their antigen-binding fragments, bispecific antibodies, or their fusion proteins) comprise one, two, and/or three heavy chain CDRs and/or one, two, and/or three light chain CDRs from: (a) an antibody designated as the 9E3 chimera (ABC1139); (b) an antibody designated as ABC1931; (c) an antibody designated as ABC1930; (d) an antibody designated as ABC1773; (e) an antibody designated as ABC1947; (f) an antibody designated as ABC2066; (g) an antibody designated as ABC2067; or (h) an antibody designated as ABC2097, as shown in Tables 6 and 7 . In some embodiments, the FAP conjugates described herein (e.g., anti-FAP antibodies or their antigen-binding fragments, bispecific antibodies, or their fusion proteins) comprise one, two, and/or three heavy chain CDRs and one, two, and/or three light chain CDRs from: (a) an antibody designated as the 9E3 chimera (ABC1139); (b) an antibody designated as ABC1931; (c) an antibody designated as ABC1930; (d) an antibody designated as ABC1773; (e) an antibody designated as ABC1947; (f) an antibody designated as ABC2066; (g) an antibody designated as ABC2067; or (h) an antibody designated as ABC2097, as shown in Tables 6 to 7 .

在一些實施例中，FAP結合劑包含重鏈可變區及輕鏈可變區。在一些實施例中，FAP結合劑包含至少一個包含重鏈可變區及重鏈恆定區之至少一部分的重鏈、及至少一個包含輕鏈可變區及輕鏈恆定區之至少一部分的輕鏈。在一些實施例中，FAP結合劑包含兩條重鏈（其中各重鏈包含重鏈可變區及重鏈恆定區之至少一部分）及兩條輕鏈（其中各輕鏈包含輕鏈可變區及輕鏈恆定區之至少一部分）。如本文中所使用，包含例如包含所有六個CDR（三個重鏈CDR及三個輕鏈CDR）之單一多肽鏈的單鏈Fv (scFv)或任何其他結合劑（例如抗體）被視為具有重鏈及輕鏈。在一些實施例中，重鏈係FAP結合劑之包含三個重鏈CDR之區。在一些實施例中，輕鏈係FAP結合劑之包含三個輕鏈CDR之區。In some embodiments, the FAP binder includes a variable region of heavy chain and a variable region of light chain. In some embodiments, the FAP binder includes at least one heavy chain including at least a portion of a variable region of heavy chain and a constant region of heavy chain, and at least one light chain including at least a portion of a variable region of light chain and a constant region of light chain. In some embodiments, the FAP binder includes two heavy chains (each heavy chain including at least a portion of a variable region of heavy chain and a constant region of heavy chain) and two light chains (each light chain including at least a portion of a variable region of light chain and a constant region of light chain). As used herein, a single-chain Fv (scFv) or any other binding agent (e.g., an antibody) comprising, for example, all six CDRs (three heavy chain CDRs and three light chain CDRs) of a single polypeptide chain is considered to have both a heavy chain and a light chain. In some embodiments, the heavy chain is the region of the FAP binding agent containing the three heavy chain CDRs. In some embodiments, the light chain is the region of the FAP binding agent containing the three light chain CDRs.

因此，在一些實施例中，FAP結合劑（例如抗FAP抗體或其抗原結合片段、雙特異性抗體、或其融合蛋白）包含本文所述之結合劑中之任一者的包含VH CDR1、VH CDR2、及/或VH CDR3的VH區及包含VL CDR1、VL CDR2、及/或VL CDR3的VL區（參見例如 表 6 至表7）。因此，在一些實施例中，本文所述之FAP結合劑（例如抗FAP抗體或其抗原結合片段、雙特異性抗體、或其融合蛋白）包含來自 表 6的一個、兩個、及/或三個重鏈CDR（例如VH CDR1、VH CDR2、及VH CDR3）及/或一個、兩個、及/或三個輕鏈CDR（例如VL CDR1、VL CDR2、及VL CDR3）。在一些實施例中，本文所述之FAP結合劑包含來自 表 7的一個、兩個、及/或三個重鏈CDR（例如VH CDR1、VH CDR2、及VH CDR3）及/或一個、兩個、及/或三個輕鏈CDR（例如VL CDR1、VL CDR2、及VL CDR3）。在一些實施例中，本文所述之FAP結合劑（例如抗FAP抗體或其抗原結合片段、雙特異性抗體、或其融合蛋白）係多特異性的（例如雙特異性的）且包含第一結合部份及第二結合部份，該第一結合部份包含來自 表 6 或表7的一個、兩個、及/或三個重鏈CDR（例如VH CDR1、VH CDR2、及VH CDR3）及/或一個、兩個、及/或三個輕鏈CDR（例如VL CDR1、VL CDR2、VL CDR3），該第二結合部份包含來自結合至非FAP的第二目標抗原之結合劑的一個、兩個、及/或三個重鏈CDR（例如VH CDR1、VH CDR2、及VH CDR3）及/或一個、兩個、及/或三個輕鏈CDR（例如VL CDR1、VL CDR2、及VL CDR3）。 Therefore, in some embodiments, the FAP conjugate (e.g., an anti-FAP antibody or its antigen-binding fragment, a bispecific antibody, or its fusion protein) comprises a VH region containing VH CDR1, VH CDR2, and/or VH CDR3 and a VL region containing VL CDR1, VL CDR2, and/or VL CDR3 of any of the conjugates described herein (see, for example, Tables 6 and 7 ). Therefore, in some embodiments, the FAP conjugates described herein (e.g., anti-FAP antibodies or their antigen-binding fragments, bispecific antibodies, or their fusion proteins) comprise one, two, and/or three heavy chain CDRs (e.g., VH CDR1, VH CDR2, and VH CDR3) and/or one, two, and/or three light chain CDRs (e.g., VL CDR1, VL CDR2, and VL CDR3) from Table 6. In some embodiments, the FAP conjugates described herein comprise one, two, and/or three heavy chain CDRs (e.g., VH CDR1, VH CDR2, and VH CDR3) and/or one, two, and/or three light chain CDRs (e.g., VL CDR1, VL CDR2, and VL CDR3) from Table 7. In some embodiments, the FAP conjugates described herein (e.g., anti-FAP antibodies or their antigen-binding fragments, bispecific antibodies, or their fusion proteins) are multispecific (e.g., bispecific) and comprise a first binding moiety and a second binding moiety. The first binding moiety comprises one, two, and/or three heavy chain CDRs (e.g., VH CDR1 , VH CDR2, and VH CDR3) and/or one, two, and/or three light chain CDRs (e.g., VL CDR1, VL CDR2, and VL CDR3) from Table 6 or Table 7. The second binding moiety comprises one, two, and/or three heavy chain CDRs (e.g., VH CDR1, VH CDR2, and VH CDR3) from a conjugate that binds to a non-FAP second target antigen. CDR3) and/or one, two, and/or three light chain CDRs (e.g., VL CDR1, VL CDR2, and VL CDR3).

在一些實施例中，本文所揭示之CDR包括衍生自相關抗體組之共有(consensus)序列（參見 表 6）。共有序列係指具有數個序列中共有的保守胺基酸及在給定胺基酸序列內變化的可變胺基酸的胺基酸序列。所提供之CDR共有序列包括對應於CDRH1、CDRH2、CDRH3、CDRL1、CDRL2、及/或CDRL3之CDR。FAP結合劑之CDR之共有序列（例如9E3嵌合體(ABC1139)至ABC2097）係顯示於 表 6中。除非另有明確指示，否則本揭露中之共有CDR已藉由Kabat編號方案識別。 In some embodiments, the CDRs disclosed herein include consensus sequences derived from the relevant antibody groups ( see Table 6 ). A consensus sequence is an amino acid sequence having a conserved amino acid common to several sequences and a variable amino acid that varies within a given amino acid sequence. The CDR consensus sequences provided include CDRs corresponding to CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and/or CDRL3. Consensus sequences of CDRs for FAP binders (e.g., 9E3 chimeras (ABC1139) to ABC2097) are shown in Table 6. Unless otherwise expressly indicated, the consensus CDRs in this disclosure have been identified using the Kabat numbering scheme.

因此，在一些實施例中，FAP結合劑包含選自下列的至少一個、兩個、三個、四個、五個、或六個CDR：(a) HCDR1，其包含IYGVN (SEQ ID NO: 26)、TAGMSVG (SEQ ID NO: 32)、GFSLSIY (SEQ ID NO: 52)、VSGFSLSIYG (SEQ ID NO: 53)、GFSLSIYG (SEQ ID NO: 54)、GFSLSTAGM (SEQ ID NO: 55)、FSGFSLSTAGMS (SEQ ID NO: 56)、或GFSLSTAGMS (SEQ ID NO: 57)的胺基酸序列；(b) HCDR2，其包含DIWWDDKKHYNPSLKD (SEQ ID NO: 33)、SGG (SEQ ID NO: 58)、IWSGGRKDYNLSLKSR (SEQ ID NO: 59)、IWSGGRK (SEQ ID NO: 60)、AIWSGGRKDYNLSLKS (SEQ ID NO: 61)、IWSGGRKDYSLSLKSR (SEQ ID NO: 62)、或AIWSGGRKDYSLSLKS (SEQ ID NO: 63)的胺基酸序列；(c) HCDR3，其包含SQDMPGYFDY (SEQ ID NO: 28)、DMIFNFYFDV (SEQ ID NO: 34)、QDMPGYFD (SEQ ID NO: 67)、SQDMPGYFD (SEQ ID NO: 68)、ARSQDMPGYFDY (SEQ ID NO: 69)、MIFNFYFD (SEQ ID NO: 70)、DMIFNFYFD (SEQ ID NO: 71)、或ARDMIFNFYFDV (SEQ ID NO: 72)的胺基酸序列；(d) LCDR1，其包含SASSRVGYMH (SEQ ID NO: 29)、NQNVDYNGNTF (SEQ ID NO: 73)、TNQNVDYNGNTF (SEQ ID NO: 74)、QNVDYNGNTF (SEQ ID NO: 75)、KTNQNVDYNGNTFMH (SEQ ID NO: 76)、TNQNVDYSGNTF (SEQ ID NO: 77)、或NQNVDYSGNTF (SEQ ID NO: 78)的胺基酸序列；(e) LCDR2，其包含LASNLAS (SEQ ID NO: 24)、DTSKLAS (SEQ ID NO: 30)、LAS (SEQ ID NO: 83)、LASNLASGIPDR (SEQ ID NO: 84)、LASNLASGIPER (SEQ ID NO: 85)、或DTS (SEQ ID NO: 86)的胺基酸序列；及(f) LCDR3，其包含QQSRNLPYT (SEQ ID NO: 25)、FQGSGYPFT (SEQ ID NO: 31)、SRNLPY (SEQ ID NO: 88)、GSGYPF (SEQ ID NO: 89)的胺基酸序列。Therefore, in some embodiments, the FAP conjugate comprises at least one, two, three, four, five, or six CDRs selected from the following: (a) HCDR1, comprising an amino acid sequence of IYGVN (SEQ ID NO: 26), TAGMSVG (SEQ ID NO: 32), GFSLSIY (SEQ ID NO: 52), VSGFSLSIYG (SEQ ID NO: 53), GFSLSIYG (SEQ ID NO: 54), GFSLSTAGM (SEQ ID NO: 55), FSGFSLSTAGMS (SEQ ID NO: 56), or GFSLSTAGMS (SEQ ID NO: 57); (b) HCDR2, comprising DIWWDDKKHYNPSLKD (SEQ ID NO: 33), SGG (SEQ ID NO: 58), IWSGGRKDYNLSLKSR (SEQ ID NO: 59), IWSGGRK (SEQ ID NO: 50), or GFSLSTAGMS (SEQ ID NO: 51). (c) HCDR3, comprising the amino acid sequences of SQDMPGYFDY (SEQ ID NO: 28), DMIFNFYFDV (SEQ ID NO: 34), QDMPGYFD (SEQ ID NO: 67), SQDMPGYFD (SEQ ID NO: 68), ARSQDMPGYFDY (SEQ ID NO: 69), MIFNFYFD (SEQ ID NO: 70), DMIFNFYFD (SEQ ID NO: 71), or ARDMIFNFYFDV (SEQ ID NO: 72); and (d) LCDR1, comprising the amino acid sequences of SASSRVGYMH (SEQ ID NO: 29), NQNVDYNGNTF (SEQ ID NO: 60), and 100, ... 73) amino acid sequences of TNQNVDYNGNTF (SEQ ID NO: 74), QNVDYNGNTF (SEQ ID NO: 75), KTNQNVDYNGNTFMH (SEQ ID NO: 76), TNQNVDYSGNTF (SEQ ID NO: 77), or NQNVDYSGNTF (SEQ ID NO: 78); (e) LCDR2 comprising amino acid sequences of LASNLAS (SEQ ID NO: 24), DTSKLAS (SEQ ID NO: 30), LAS (SEQ ID NO: 83), LASNLASGIPDR (SEQ ID NO: 84), LASNLASGIPER (SEQ ID NO: 85), or DTS (SEQ ID NO: 86); and (f) LCDR3 comprising amino acid sequences of QQSRNLPYT (SEQ ID NO: 25), FQGSGYPFT (SEQ ID NO: 31), SRNLPY (SEQ ID NO: 78), or DTS (SEQ ID NO: 79). 88), the amino acid sequence of GSGYPF (SEQ ID NO: 89).

在一些實施例中，本文所述之FAP結合劑（例如抗FAP抗體或其抗原結合片段、雙特異性抗體、或其融合蛋白）包含來自下列的一個、兩個、及/或三個重鏈CDR及/或一個、兩個、及/或三個輕鏈CDR：(a)指定為9E3嵌合體(ABC1139)的抗體；(b)指定為ABC1931的抗體；(c)指定為ABC1930的抗體；(d)指定為ABC1773的抗體；(e)指定為ABC1947的抗體；(f)指定為ABC2066的抗體；(g)指定為ABC2067的抗體；或(h)指定為ABC2097的抗體，如 表 3中所示。 In some embodiments, the FAP conjugates described herein (e.g., anti-FAP antibodies or their antigen-binding fragments, bispecific antibodies, or their fusion proteins) comprise one, two, and/or three heavy chain CDRs and/or one, two, and/or three light chain CDRs from: (a) an antibody designated as the 9E3 chimera (ABC1139); (b) an antibody designated as ABC1931; (c) an antibody designated as ABC1930; (d) an antibody designated as ABC1773; (e) an antibody designated as ABC1947; (f) an antibody designated as ABC2066; (g) an antibody designated as ABC2067; or (h) an antibody designated as ABC2097, as shown in Table 3 .

在一些實施例中，本文提供之六個CDR中之任一者可作為次部分與本文提供之其他CDR中之任一者組合以在構築體中組合為總共六個CDR。因此，在一些實施例中，來自第一抗體之兩個CDR（例如HCDR1及HCDR2）可與來自第二抗體之四個CDR（HCDR3、LCDR1、LCDR2、及LCDR3）組合。在一些實施例中，CDR中之一或多者的二或更少個胺基酸殘基可經修飾以獲得其變體或衍生物。在一些實施例中，CDR中之1、2、3、4、5、或6者的二或更少個胺基酸殘基可經修飾。In some embodiments, any one of the six CDRs provided herein may be combined as a sub-part with any of the other CDRs provided herein to form a total of six CDRs in the structure. Thus, in some embodiments, two CDRs from the first antibody (e.g., HCDR1 and HCDR2) may be combined with four CDRs from the second antibody (HCDR3, LCDR1, LCDR2, and LCDR3). In some embodiments, two or fewer amino acid residues of one or more of the CDRs may be modified to obtain variants or derivatives thereof. In some embodiments, two or fewer amino acid residues of CDRs 1, 2, 3, 4, 5, or 6 may be modified.

在一些實施例中，FAP結合劑（包括人類FAP結合劑）的CDR可根據任何適合的編號系統來判定。本文中所使用的編號系統的實例係Kabat、IMGT、Honegger、及Chothia，然而，所屬技術領域中具有通常知識者將容易理解，沿著本文所述之包括人類FAP結合劑之FAP結合劑（例如抗FAP抗體或其抗原結合片段、雙特異性抗體、或其融合蛋白）的VH（例如CDR1、CDR2、或CDR3）及/或VL（例如CDR1、CDR2、或CDR3）區之一或多個CDR的位置可變化一個、兩個、三個、四個、五個、或六個胺基酸位置，只要與FAP（例如人類FAP）的結合得以維持（例如，實質上維持，例如至少50%、至少60%、至少70%、至少80%、至少90%、至少95%）。例如，在一些實施例中，定義如 表 3所述之CDR的位置可藉由相對於目前CDR位置將CDR的N端及/或C端邊界移位一個、兩個、三個、四個、五個、或六個胺基酸來變化，只要與FAP（例如人類FAP）的結合得以維持（例如，實質上維持，例如至少50%、至少60%、至少70%、至少80%、至少90%、至少95%）。在其他實施例中，沿著本文所述之包括人類FAP結合劑之FAP結合劑（例如抗FAP抗體或其抗原結合片段、雙特異性抗體、或其融合蛋白）的VH（例如CDR1、CDR2、或CDR3）及/或VL（例如CDR1、CDR2、或CDR3）區之一或多個CDR的長度可變化（例如，變得更短或更長）一個、兩個、三個、四個、五個、或更多個胺基酸，只要與FAP（例如人類FAP）的結合得以維持（例如，實質上維持，例如至少50%、至少60%、至少70%、至少80%、至少90%、至少95%）。例如，在一些實施例中，本文所述之VH及/或VL CDR1、CDR2、及/或CDR3可比由 表 6 及表7所述之序列中之任一者描述之CDR中的一或多者短一個、兩個、三個、四個、五個、或更多個胺基酸，只要與FAP（例如人類FAP）的結合得以維持（例如，實質上維持，例如至少50%、至少60%、至少70%、至少80%、至少90%、至少95%）。在其他實施例中，本文所述之VH及/或VL CDR1、CDR2、及/或CDR3可比由 表 6 及表7所述之序列中之任一者描述之CDR中的一或多者長一個、兩個、三個、四個、五個、或更多個胺基酸，只要與FAP（例如人類FAP）的結合得以維持（例如，實質上維持，例如至少50%、至少60%、至少70%、至少80%、至少90%、至少95%）。在其他實施例中，本文所述之VH及/或VL CDR1、CDR2、及/或CDR3的胺基端與由 表 6 及表7所述之序列中之任一者描述之CDR中的一或多者相比可延伸一個、兩個、三個、四個、五個、或更多個胺基酸，只要與FAP（例如人類FAP）的結合得以維持（例如，實質上維持，例如至少50%、至少60%、至少70%、至少80%、至少90%、至少95%）。在其他實施例中，本文所述之VH及/或VL CDR1、CDR2、及/或CDR3的羧基端與由 表 6 及表7所述之序列中之任一者描述之CDR中的一或多者相比可延伸一個、兩個、三個、四個、五個、或更多個胺基酸，只要與FAP（例如人類FAP）的結合得以維持（例如，實質上維持，例如至少50%、至少60%、至少70%、至少80%、至少90%、至少95%）。在其他實施例中，本文所述之VH及/或VL CDR1、CDR2、及/或CDR3的胺基端與由 表 6 及表7所述之序列中之任一者描述之CDR中的一或多者相比可縮短一個、兩個、三個、四個、五個、或更多個胺基酸，只要與FAP（例如人類FAP）的結合得以維持（例如，實質上維持，例如至少50%、至少60%、至少70%、至少80%、至少90%、至少95%）。在一些實施例中，本文所述之VH及/或VL CDR1、CDR2、及/或CDR3的羧基端與由 表 6 及表7所述之序列中之任一者描述之CDR中的一或多者相比可縮短一個、兩個、三個、四個、五個、或更多個胺基酸，只要與FAP（例如人類FAP）的結合得以維持（例如，實質上維持，例如至少50%、至少60%、至少70%、至少80%、至少90%、至少95%）。可使用所屬技術領域中已知的任何方法來確定是否維持與FAP（例如人類FAP）的結合，例如，本文所述之「實例」部分中所述的結合檢定及條件。例如，本文所述之 實例 2描述了用於測量與FAP（例如人類FAP）的結合的檢定。 In some implementations, the CDR of FAP binders (including human FAP binders) can be determined according to any suitable numbering system. Examples of the numbering systems used herein are Kabat, IMGT, Honegger, and Chothia. However, it will be readily understood by those skilled in the art that the positions of one or more CDRs along the VH (e.g., CDR1, CDR2, or CDR3) and/or VL (e.g., CDR1, CDR2, or CDR3) regions of an FAP binder (e.g., an anti-FAP antibody or its antigen-binding fragment, a bispecific antibody, or its fusion protein) described herein may vary by one, two, three, four, five, or six amino acid positions, provided that the binding to FAP (e.g., human FAP) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). For example, in some embodiments, the position of the CDR as defined in Table 3 can be varied by shifting the N-terminal and/or C-terminal boundary of the CDR by one, two, three, four, five, or six amino acids relative to the current CDR position, as long as the binding to FAP (e.g., human FAP) is maintained (e.g., substantially maintained, such as at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). In other embodiments, the length of one or more CDRs along the VH (e.g., CDR1, CDR2, or CDR3) and/or VL (e.g., CDR1, CDR2, or CDR3) regions of an FAP binder (e.g., an anti-FAP antibody or its antigen-binding fragment, a bispecific antibody, or its fusion protein) described herein may vary (e.g., become shorter or longer) by one, two, three, four, five, or more amino acids, provided that the binding to FAP (e.g., human FAP) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). For example, in some embodiments, the VH and/or VL CDR1, CDR2, and/or CDR3 described herein may be one, two, three, four, five, or more amino acids shorter than one or more of the CDRs described by any of the sequences described in Tables 6 and 7 , provided that the binding to FAP (e.g., human FAP) is maintained (e.g., substantially maintained, such as at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). In other embodiments, the VH and/or VL CDR1, CDR2, and/or CDR3 described herein may be longer than one, two, three, four, five, or more amino acids than one or more of the CDRs described by any of the sequences described in Tables 6 and 7 , provided that the binding to FAP (e.g., human FAP) is maintained (e.g., substantially maintained, such as at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). In other embodiments, the amino termini of the VH and/or VL CDR1, CDR2, and/or CDR3 described herein may be extended by one, two, three, four, five, or more amino acids compared to one or more of the CDRs described in any of the sequences described in Tables 6 and 7 , provided that the binding to FAP (e.g., human FAP) is maintained (e.g., substantially maintained, such as at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). In other embodiments, the carboxyl terminus of the VH and/or VL CDR1, CDR2, and/or CDR3 described herein may be extended by one, two, three, four, five, or more amino acids compared to one or more of the CDRs described in any of the sequences described in Tables 6 and 7 , provided that the binding to FAP (e.g., human FAP) is maintained (e.g., substantially maintained, such as at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). In other embodiments, the amino termini of the VH and/or VL CDR1, CDR2, and/or CDR3 described herein may be shortened by one, two, three, four, five, or more amino acids compared to one or more of the CDRs described in any of the sequences described in Tables 6 and 7 , provided that the binding to FAP (e.g., human FAP) is maintained (e.g., substantially maintained, such as at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). In some embodiments, the carboxyl termini of the VH and/or VL CDR1, CDR2, and/or CDR3 described herein may be shortened by one, two, three, four, five, or more amino acids compared to one or more of the CDRs described in any of the sequences described in Tables 6 and 7 , provided that binding to FAP (e.g., human FAP) is maintained (e.g., substantially maintained, such as at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). Any method known in the art can be used to determine whether binding to FAP (e.g., human FAP) is maintained, for example, the binding tests and conditions described in the "Examples" section herein. For example, Example 2 described herein describes a test for measuring binding to FAP (e.g., human FAP).

在一些實施例中，本文所述之包括人類FAP結合劑的FAP結合劑（例如抗體，諸如雙特異性抗體）包含VH區或VH域。在其他實施例中，本文所述之包括人類FAP結合劑的FAP結合劑例如抗體，諸如雙特異性抗體）包含VL區或VL域。在一些實施例中，本文所述之包括人類FAP結合劑的FAP結合劑（例如抗體，諸如雙特異性抗體）具有下列的組合：(i) VH域或VH區；及/或(ii) VL域或VL區。In some embodiments, the FAP binder comprising a human FAP binder (e.g., an antibody, such as a bispecific antibody) described herein comprises a VH region or VH domain. In other embodiments, the FAP binder comprising a human FAP binder described herein (e.g., an antibody, such as a bispecific antibody) comprises a VL region or VL domain. In some embodiments, the FAP binder comprising a human FAP binder described herein (e.g., an antibody, such as a bispecific antibody) has the following combination: (i) a VH domain or VH region; and/or (ii) a VL domain or VL region.

在一些實施例中，FAP結合劑包含與SEQ ID NO: 35、37、或41之胺基酸序列具有至少90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、或100%序列同一性的重鏈可變域(VH)序列。在一些實施例中，具有至少90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%同一性的VH序列相對於參考序列含有胺基酸修飾，諸如取代（例如保守性取代）、插入、或缺失，但包含該序列的FAP結合劑保留結合至FAP的能力。在一些實施例中，總共1至10個胺基酸（例如1、2、3、4、5、6、7、8、9、或10個胺基酸）在SEQ ID NO: 35、37、或41中已經修飾（例如取代、插入、及/或缺失）。在一些實施例中，修飾（例如取代、插入、或缺失）出現在CDR之外的區中（亦即，在FR中）。可選地，FAP結合劑包含SEQ ID NO: 35、37、或41中之VH序列，包括該序列之轉譯後修飾。在一些實施例中，此類FAP結合劑包含：(a) HCDR1，其包含IYGVN (SEQ ID NO: 26)、TAGMSVG (SEQ ID NO: 32)、GFSLSIY (SEQ ID NO: 52)、VSGFSLSIYG (SEQ ID NO: 53)、GFSLSIYG (SEQ ID NO: 54)、GFSLSTAGM (SEQ ID NO: 55)、FSGFSLSTAGMS (SEQ ID NO: 56)、或GFSLSTAGMS (SEQ ID NO: 57)的胺基酸序列；(b) HCDR2，其包含DIWWDDKKHYNPSLKD (SEQ ID NO: 33)、SGG (SEQ ID NO: 58)、IWSGGRKDYNLSLKSR (SEQ ID NO: 59)、IWSGGRK (SEQ ID NO: 60)、AIWSGGRKDYNLSLKS (SEQ ID NO: 61)、IWSGGRKDYSLSLKSR (SEQ ID NO: 62)、或AIWSGGRKDYSLSLKS (SEQ ID NO: 63)的胺基酸序列；(c) HCDR3，其包含SQDMPGYFDY (SEQ ID NO: 28)、DMIFNFYFDV (SEQ ID NO: 34)、QDMPGYFD (SEQ ID NO: 67)、SQDMPGYFD (SEQ ID NO: 68)、ARSQDMPGYFDY (SEQ ID NO: 69)、MIFNFYFD (SEQ ID NO: 70)、DMIFNFYFD (SEQ ID NO: 71)、或ARDMIFNFYFDV (SEQ ID NO: 72)的胺基酸序列。In some embodiments, the FAP binder comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 35, 37, or 41. In some embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains amino acid modifications relative to the reference sequence, such as substitution (e.g., conservative substitution), insertion, or deletion, but the FAP binder comprising this sequence retains its ability to bind to FAP. In some embodiments, a total of 1 to 10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) have been modified (e.g., substituted, inserted, and/or deleted) in SEQ ID NO: 35, 37, or 41. In some embodiments, the modification (e.g., substitution, insertion, or deletion) occurs in a region outside the CDR (i.e., in the FR). Alternatively, the FAP binder comprises the VH sequence in SEQ ID NO: 35, 37, or 41, including post-translational modifications of that sequence. In some embodiments, such FAP conjugates comprise: (a) HCDR1 comprising an amino acid sequence comprising IYGVN (SEQ ID NO: 26), TAGMSVG (SEQ ID NO: 32), GFSLSIY (SEQ ID NO: 52), VSGFSLSIYG (SEQ ID NO: 53), GFSLSIYG (SEQ ID NO: 54), GFSLSTAGM (SEQ ID NO: 55), FSGFSLSTAGMS (SEQ ID NO: 56), or GFSLSTAGMS (SEQ ID NO: 57); and (b) HCDR2 comprising DIWWDDKKHYNPSLKD (SEQ ID NO: 33), SGG (SEQ ID NO: 58), IWSGGRKDYNLSLKSR (SEQ ID NO: 59), IWSGGRK (SEQ ID NO: 60), or AIWSGGRKDYNLSLKS (SEQ ID NO: 57). 61) The amino acid sequence of IWSGGRKDYSLSLKSR (SEQ ID NO: 62) or AIWSGGRKDYSLSLKS (SEQ ID NO: 63); (c) HCDR3, which contains the amino acid sequence of SQDMPGYFDY (SEQ ID NO: 28), DMIFNFYFDV (SEQ ID NO: 34), QDMPGYFD (SEQ ID NO: 67), SQDMPGYFD (SEQ ID NO: 68), ARSQDMPGYFDY (SEQ ID NO: 69), MIFNFYFD (SEQ ID NO: 70), DMIFNFYFD (SEQ ID NO: 71), or ARDMIFNFYFDV (SEQ ID NO: 72).

在一些實施例中，提供一種FAP結合劑，其中抗體包含與SEQ ID NO: 36、38、或42之胺基酸序列具有至少90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、或100%序列同一性的輕鏈可變域(VL)。在一些實施例中，具有至少90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%同一性的VL序列相對於參考序列含有胺基酸修飾，諸如取代（例如保守性取代）、插入、或缺失，但包含該序列的FAP結合劑保留結合至FAP的能力。在一些實施例中，總共1至10個胺基酸（例如1、2、3、4、5、6、7、8、9、或10個胺基酸）在SEQ ID NO: 36、38、或42中已經修飾（例如取代、插入、及/或缺失）。在一些實施例中，修飾（例如取代、插入、或缺失）出現在CDR之外的區中（亦即，在FR中）。可選地，FAP結合劑包含SEQ ID NO: 36、38、或42中之VL序列，包括該序列之轉譯後修飾。在一些實施例中，此類FAP結合劑包含(d) LCDR1，其包含SASSRVGYMH (SEQ ID NO: 29)、NQNVDYNGNTF (SEQ ID NO: 73)、TNQNVDYNGNTF (SEQ ID NO: 74)、QNVDYNGNTF (SEQ ID NO: 75)、KTNQNVDYNGNTFMH (SEQ ID NO: 76)、TNQNVDYSGNTF (SEQ ID NO: 77)、或NQNVDYSGNTF (SEQ ID NO: 78)的胺基酸序列；(e) LCDR2，其包含LASNLAS (SEQ ID NO: 24)、DTSKLAS (SEQ ID NO: 30)、LAS (SEQ ID NO: 83)、LASNLASGIPDR (SEQ ID NO: 84)、LASNLASGIPER (SEQ ID NO: 85)、或DTS (SEQ ID NO: 86)的胺基酸序列；及(f) LCDR3，其包含QQSRNLPYT (SEQ ID NO: 25)、FQGSGYPFT (SEQ ID NO: 31)、SRNLPY (SEQ ID NO: 88)、GSGYPF (SEQ ID NO: 89)的胺基酸序列。In some embodiments, an FAP binder is provided, wherein the antibody comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 36, 38, or 42. In some embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains an amino acid modification relative to the reference sequence, such as substitution (e.g., conservative substitution), insertion, or deletion, but the FAP binder comprising this sequence retains the ability to bind to FAP. In some embodiments, a total of 1 to 10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) have been modified (e.g., substituted, inserted, and/or deleted) in SEQ ID NO: 36, 38, or 42. In some embodiments, the modification (e.g., substitution, insertion, or deletion) occurs in a region outside the CDR (i.e., in the FR). Alternatively, the FAP binder comprises the VL sequence in SEQ ID NO: 36, 38, or 42, including post-translational modifications of that sequence. In some embodiments, such FAP conjugates comprise (d) LCDR1, which comprises the amino acid sequence SASSRVGYMH (SEQ ID NO: 29), NQNVDYNGNTF (SEQ ID NO: 73), TNQNVDYNGNTF (SEQ ID NO: 74), QNVDYNGNTF (SEQ ID NO: 75), KTNQNVDYNGNTFMH (SEQ ID NO: 76), TNQNVDYSGNTF (SEQ ID NO: 77), or NQNVDYSGNTF (SEQ ID NO: 78); and (e) LCDR2, which comprises the amino acid sequence LASNLAS (SEQ ID NO: 24), DTSKLAS (SEQ ID NO: 30), LAS (SEQ ID NO: 83), LASNLASGIPDR (SEQ ID NO: 84), LASNLASGIPER (SEQ ID NO: 85), or DTS (SEQ ID NO: 86). (f) an amino acid sequence comprising QQSRNLPYT (SEQ ID NO: 25), FQGSGYPFT (SEQ ID NO: 31), SRNLPY (SEQ ID NO: 88), and GSGYPF (SEQ ID NO: 89).

在一些實施例中，本文所提供之VH可作為FAP結合劑的次部分與本文所提供之一種VL中之任一者組合，以在構築體的次部分中組合為總共有一個VH及一個VL，及在構築體中組合為2個VH及2個VL。因此，在一些實施例中，來自第一抗體的VH可與來自第二抗體的VL組合。In some embodiments, the VH provided herein can be combined as a sub-part of the FAP binder with any of the VLs provided herein, to form a total of one VH and one VL in the sub-part of the structure, and to form two VHs and two VLs in the structure. Therefore, in some embodiments, the VH from the first antibody can be combined with the VL from the second antibody.

在一些實施例中，FAP結合劑包含與SEQ ID NO: 35、37、或41之胺基酸序列具有至少90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、或100%序列同一性的重鏈可變域(VH)序列及與SEQ ID NO: 36、38、或42之胺基酸序列具有至少90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、或100%序列同一性的輕鏈可變域(VL)。在一些實施例中，相對於參考序列具有至少90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%同一性的VH序列含有修飾，諸如取代（例如保守性取代）、插入、或缺失，且相對於參考序列具有至少90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%同一性的VL序列含有修飾，諸如取代（例如保守性取代）、插入、或缺失，但包含該序列的FAP結合劑保留結合至FAP的能力。在一些實施例中，總共1至10個胺基酸（例如1、2、3、4、5、6、7、8、9、或10個胺基酸）在SEQ ID NO: 35、37、或41中已經修飾（例如取代、插入、及/或缺失）；及總共1至10個胺基酸（例如1、2、3、4、5、6、7、8、9、或10個胺基酸）在SEQ ID NO: 36、38、或42中已經修飾（例如取代、插入、及/或缺失）。在一些實施例中，修飾（例如取代、插入、或缺失）出現在CDR之外的區中（亦即，在FR中）。在一些實施例中，此類FAP結合劑包含：(a) HCDR1，其包含IYGVN (SEQ ID NO: 26)、TAGMSVG (SEQ ID NO: 32)、GFSLSIY (SEQ ID NO: 52)、VSGFSLSIYG (SEQ ID NO: 53)、GFSLSIYG (SEQ ID NO: 54)、GFSLSTAGM (SEQ ID NO: 55)、FSGFSLSTAGMS (SEQ ID NO: 56)、或GFSLSTAGMS (SEQ ID NO: 57)的胺基酸序列；(b) HCDR2，其包含DIWWDDKKHYNPSLKD (SEQ ID NO: 33)、SGG (SEQ ID NO: 58)、IWSGGRKDYNLSLKSR (SEQ ID NO: 59)、IWSGGRK (SEQ ID NO: 60)、AIWSGGRKDYNLSLKS (SEQ ID NO: 61)、IWSGGRKDYSLSLKSR (SEQ ID NO: 62)、或AIWSGGRKDYSLSLKS (SEQ ID NO: 63)的胺基酸序列；(c) HCDR3，其包含SQDMPGYFDY (SEQ ID NO: 28)、DMIFNFYFDV (SEQ ID NO: 34)、QDMPGYFD (SEQ ID NO: 67)、SQDMPGYFD (SEQ ID NO: 68)、ARSQDMPGYFDY (SEQ ID NO: 69)、MIFNFYFD (SEQ ID NO: 70)、DMIFNFYFD (SEQ ID NO: 71)、或ARDMIFNFYFDV (SEQ ID NO: 72)的胺基酸序列；(d) LCDR1，其包含SASSRVGYMH (SEQ ID NO: 29)、NQNVDYNGNTF (SEQ ID NO: 73)、TNQNVDYNGNTF (SEQ ID NO: 74)、QNVDYNGNTF (SEQ ID NO: 75)、KTNQNVDYNGNTFMH (SEQ ID NO: 76)、TNQNVDYSGNTF (SEQ ID NO: 77)、或NQNVDYSGNTF (SEQ ID NO: 78)的胺基酸序列；(e) LCDR2，其包含LASNLAS (SEQ ID NO: 24)、DTSKLAS (SEQ ID NO: 30)、LAS (SEQ ID NO: 83)、LASNLASGIPDR (SEQ ID NO: 84)、LASNLASGIPER (SEQ ID NO: 85)、或DTS (SEQ ID NO: 86)的胺基酸序列；及(f) LCDR3，其包含QQSRNLPYT (SEQ ID NO: 25)、FQGSGYPFT (SEQ ID NO: 31)、SRNLPY (SEQ ID NO: 88)、GSGYPF (SEQ ID NO: 89)的胺基酸序列。In some embodiments, the FAP conjugate comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 35, 37, or 41, and a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 36, 38, or 42. In some embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the reference sequence contains modifications such as substitution (e.g., conservative substitution), insertion, or deletion, and the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the reference sequence contains modifications such as substitution (e.g., conservative substitution), insertion, or deletion, but the FAP binder containing the sequence retains the ability to bind to FAP. In some embodiments, a total of 1 to 10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) have been modified (e.g., substituted, inserted, and/or deleted) in SEQ ID NO: 35, 37, or 41; and a total of 1 to 10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) have been modified (e.g., substituted, inserted, and/or deleted) in SEQ ID NO: 36, 38, or 42. In some embodiments, the modifications (e.g., substitution, insertion, or deletion) occur in regions outside the CDR (i.e., in the FR). In some embodiments, such FAP conjugates comprise: (a) HCDR1 comprising an amino acid sequence comprising IYGVN (SEQ ID NO: 26), TAGMSVG (SEQ ID NO: 32), GFSLSIY (SEQ ID NO: 52), VSGFSLSIYG (SEQ ID NO: 53), GFSLSIYG (SEQ ID NO: 54), GFSLSTAGM (SEQ ID NO: 55), FSGFSLSTAGMS (SEQ ID NO: 56), or GFSLSTAGMS (SEQ ID NO: 57); and (b) HCDR2 comprising DIWWDDKKHYNPSLKD (SEQ ID NO: 33), SGG (SEQ ID NO: 58), IWSGGRKDYNLSLKSR (SEQ ID NO: 59), IWSGGRK (SEQ ID NO: 60), or AIWSGGRKDYNLSLKS (SEQ ID NO: 57). (c) HCDR3, comprising the amino acid sequences of SQDMPGYFDY (SEQ ID NO: 28), DMIFNFYFDV (SEQ ID NO: 34), QDMPGYFD (SEQ ID NO: 67), SQDMPGYFD (SEQ ID NO: 68), ARSQDMPGYFDY (SEQ ID NO: 69), MIFNFYFD (SEQ ID NO: 70), DMIFNFYFD (SEQ ID NO: 71), or ARDMIFNFYFDV (SEQ ID NO: 72); (d) LCDR1, comprising the amino acid sequences of SASSRVGYMH (SEQ ID NO: 29), NQNVDYNGNTF (SEQ ID NO: 73), TNQNVDYNGNTF (SEQ ID NO: 74), and RNMIFNFYFDV (SEQ ID NO: 75). 74) The amino acid sequence of QNVDYNGNTF (SEQ ID NO: 75), KTNQNVDYNGNTFMH (SEQ ID NO: 76), TNQNVDYSGNTF (SEQ ID NO: 77), or NQNVDYSGNTF (SEQ ID NO: 78); (e) LCDR2, comprising the amino acid sequence of LASNLAS (SEQ ID NO: 24), DTSKLAS (SEQ ID NO: 30), LAS (SEQ ID NO: 83), LASNLASGIPDR (SEQ ID NO: 84), LASNLASGIPER (SEQ ID NO: 85), or DTS (SEQ ID NO: 86); and (f) LCDR3, comprising the amino acid sequence of QQSRNLPYT (SEQ ID NO: 25), FQGSGYPFT (SEQ ID NO: 31), SRNLPY (SEQ ID NO: 88), GSGYPF (SEQ ID NO: 78), or DTS (SEQ ID NO: 79). The amino acid sequence of 89).

在一些實施例中，提供一種FAP結合劑，其中抗體包含與SEQ ID NO: 1、4-6、或8-16之胺基酸序列具有至少90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、或100%序列同一性的重鏈(HC)。可選地，FAP結合劑包含SEQ ID NO: 1、4-6、或8-16中之HC序列，包括轉譯後修飾。In some embodiments, an FAP conjugate is provided, wherein the antibody comprises a heavy chain (HC) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 1, 4-6, or 8-16. Optionally, the FAP conjugate comprises the HC sequence of SEQ ID NO: 1, 4-6, or 8-16, including post-translational modifications.

在一些實施例中，提供一種FAP結合劑，其中抗體包含與SEQ ID NO: 5或6之胺基酸序列具有至少90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、或100%序列同一性的重鏈(HC)。可選地，FAP結合劑包含SEQ ID NO: 5或6中之HC序列，包括轉譯後修飾。In some embodiments, an FAP conjugate is provided, wherein the antibody comprises a heavy chain (HC) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 5 or 6. Optionally, the FAP conjugate comprises the HC sequence of SEQ ID NO: 5 or 6, including post-translational modifications.

在一些實施例中，提供一種FAP結合劑，其中抗體包含與SEQ ID NO: 2、3、或7之胺基酸序列具有至少90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、或100%序列同一性的LC。可選地，FAP結合劑包含SEQ ID NO: 2、3、或7中之LC序列，包括轉譯後修飾。In some embodiments, an FAP conjugate is provided, wherein the antibody comprises an LC having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 2, 3, or 7. Optionally, the FAP conjugate comprises the LC sequence of SEQ ID NO: 2, 3, or 7, including post-translational modifications.

在一些實施例中，提供一種FAP結合劑，其中抗體包含與SEQ ID NO: 3之胺基酸序列具有至少90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、或100%序列同一性的LC。可選地，FAP結合劑包含SEQ ID NO: 3中之LC序列，包括轉譯後修飾。In some embodiments, an FAP binder is provided, wherein the antibody comprises an LC having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 3. Optionally, the FAP binder comprises the LC sequence of SEQ ID NO: 3, including post-translational modifications.

在一些實施例中，FAP結合劑包含如本文所提供之實施例中之任一者的HC、及如本文所提供之實施例中之任一者的LC。In some embodiments, the FAP binder includes HC of any of the embodiments provided herein and LC of any of the embodiments provided herein.

在一些實施例中，提供一種FAP結合劑，其中抗體包含與SEQ ID NO: 1、4-6、或8-16之胺基酸序列具有至少90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、或100%序列同一性的重鏈(HC)及與SEQ ID NO: 2、3或7之胺基酸序列具有至少90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、或100%序列同一性的LC，包括該等序列之轉譯後修飾。In some embodiments, an FAP conjugate is provided, wherein the antibody comprises a heavy chain (HC) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 1, 4-6, or 8-16, and an LC having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 2, 3, or 7, including post-translational modifications of such sequences.

在一些實施例中，提供一種FAP結合劑，其中抗體包含與SEQ ID NO: 5或6之胺基酸序列具有至少90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、或100%序列同一性的重鏈(HC)及與SEQ ID NO: 3之胺基酸序列具有至少90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、或100%序列同一性的LC，包括該等序列之轉譯後修飾。In some embodiments, an FAP binder is provided, wherein the antibody comprises a heavy chain (HC) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 5 or 6, and an LC having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 3, including post-translational modifications of such sequences.

在一些實施例中，提供一種FAP結合劑，其中抗體包含與SEQ ID NO: 5之胺基酸序列具有至少90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、或100%序列同一性的第一重鏈(HC1)、與SEQ ID NO: 6之胺基酸序列具有至少90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、或100%序列同一性的第二重鏈(HC2)、及與SEQ ID NO: 3之胺基酸序列具有至少90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、或100%序列同一性的LC，包括該等序列之轉譯後修飾。In some embodiments, an FAP binder is provided, wherein the antibody comprises a first heavy chain (HC1) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 5, a second heavy chain (HC2) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 6, and an LC having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 3, including post-translational modifications of such sequences.

本文所提供之FAP結合劑可係抗FAP抗體或其抗原結合片段、其雙特異性抗體、或其融合蛋白。應理解，提及抗體FAP結合劑時，亦指靶向其或其抗原結合片段的抗體、其雙特異性抗體、或包含其的融合蛋白，且反之亦然。因此，本文提供抗FAP抗體及其片段。 抗FAP抗體或其抗原結合片段 The FAP conjugates described herein may be anti-FAP antibodies or their antigen-binding fragments, their bispecific antibodies, or their fusion proteins. It should be understood that when referring to antibody FAP conjugates, it also refers to antibodies targeting them or their antigen-binding fragments, their bispecific antibodies, or fusion proteins containing them, and vice versa. Therefore, this article provides anti-FAP antibodies and their fragments. Anti-FAP antibodies or their antigen-binding fragments

在一些實施例中，本文所提供之FAP結合劑係抗FAP抗體或其功能片段。因此，本文提供抗FAP抗體、其片段、或其用途。在一些實施例中，本文提供一種經單離抗FAP抗體。在一些實施例中，本文提供一種經單離抗FAP抗體片段。在一些實施例中，本文所提供之抗FAP抗體可用於治療及/或診斷方法，諸如偵測樣本中的FAP的方法、治療方法、診斷方法、及/或預後方法。本文進一步描述診斷及治療方法。應理解，本文提及抗體時，亦提及其功能片段（例如單鏈抗體、經單離抗體高度變異域、其結合片段）及其經修飾變異體，包括其衍生物（例如與受質或與蛋白質或蛋白質-配體對的配體接合的抗體）。In some embodiments, the FAP binders provided herein are anti-FAP antibodies or functional fragments thereof. Therefore, this document provides anti-FAP antibodies, their fragments, or their uses. In some embodiments, this document provides a transisocyanate anti-FAP antibody. In some embodiments, this document provides a transisocyanate anti-FAP antibody fragment. In some embodiments, the anti-FAP antibodies provided herein can be used for treatment and/or diagnostic methods, such as methods for detecting FAP in samples, treatment methods, diagnostic methods, and/or prognostic methods. Diagnostic and treatment methods are further described herein. It should be understood that when referring to antibodies in this article, we also refer to their functional segments (e.g., single-chain antibodies, highly variable domains of single-chain antibodies, their binding segments) and their modified variants, including their derivatives (e.g., antibodies that bind to receptors or ligands of proteins or protein-ligand pairs).

在一些實施例中，本文所描述的抗體包括免疫球蛋白分子及免疫球蛋白分子的免疫活性部分，包括含有結合至FAP抗原之一或多個抗原結合位點的分子。In some embodiments, the antibodies described herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, including molecules containing one or more antigen-binding sites to FAP antigens.

抗體可係任何類型（例如IgG、IgE、IgM、IgD、IgA、或IgY）、任何類別、（例如IgG1、IgG2、IgG3、IgG4、IgA1、或IgA2）、或任何子類（例如IgG2a或IgG2b）的免疫球蛋白分子。在一些實施例中，本文所述之抗體係IgG抗體（例如人類IgG），或其一類別（例如人類IgG1、IgG2、IgG3、或IgG4）或子類。Antibodies can be any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subclass (e.g., IgG2a or IgG2b) of immunoglobulin molecules. In some embodiments, the antibodies described herein are IgG antibodies (e.g., human IgG), or a class (e.g., human IgG1, IgG2, IgG3, or IgG4) or subclass thereof.

在一些實施例中，抗體係包含兩個重(H)鏈/輕(L)鏈對的4鏈抗體單元，其中H鏈的胺基酸序列係相同或不相同的，且L鏈的胺基酸序列係相同的。在一些實施例中，H鏈的胺基酸序列係不相同的。本文進一步描述產生不相同的重鏈胺基酸序列的重鏈胺基酸序列的修飾，例如鈕扣突變。In some embodiments, the antibody system comprises a quadruple-strand antibody unit consisting of two heavy (H)-chain/light (L)-chain pairs, wherein the amino acid sequences of the H-chain are identical or different, and the amino acid sequences of the L-chain are identical. In some embodiments, the amino acid sequences of the H-chain are different. This document further describes modifications to the heavy-chain amino acid sequences that produce different heavy-chain amino acid sequences, such as button mutations.

在一些實施例中，H鏈及L鏈包含恆定區，例如人類恆定區。在一些實施例中，此類抗體之L鏈恆定區係κ或λ輕鏈恆定區，例如人類κ或λ輕鏈恆定區。在一些實施例中，此類抗體之H鏈恆定區包含γ重鏈恆定區，例如人類γ重鏈恆定區。在一些實施例中，此類抗體包含IgG恆定區，例如人類IgG恆定區（例如IgG1、IgG2、IgG3、及/或IgG4恆定區）。In some embodiments, the H-chain and L-chain contain constant regions, such as human constant regions. In some embodiments, the constant region of the L-chain of this type of antibody is a constant region of the κ or λ light chain, such as a constant region of the human κ or λ light chain. In some embodiments, the constant region of the H-chain of this type of antibody contains a constant region of the γ heavy chain, such as a constant region of the human γ heavy chain. In some embodiments, this type of antibody contains a constant region of IgG, such as a constant region of human IgG (e.g., constant regions of IgG1, IgG2, IgG3, and/or IgG4).

本文所述之抗體及其片段包括但不限於合成抗體、單株抗體、重組產生的抗體、多特異性抗體（例如，包括雙特異性抗體）、人類抗體、人源化抗體、嵌合抗體、胞內抗體、單鏈Fv (single-chain Fv, scFv)（例如，包括單特異性、雙特異性等）、駱駝化抗體、Fab片段、F(ab')片段、二硫鍵連接的Fv (sdFv)、抗特應（抗Id）抗體、Fab串聯lg (FIT-lg)；DVD-lg；雜合融合瘤（四源雜交瘤（quadroma或tetradoma）)；抗運載蛋白平台(Pieris)；雙鏈抗體(diabody)；單鏈雙鏈抗體(single chain diabody)；串聯單鏈Fv片段；TandAb、三特異性Ab (Affimed)；Darts雙親和力重靶向(Macrogenics)；雙特異性Xmab (Xencor)；雙特異性T細胞接合器(BiTE; Amgen; 55kDa)；三抗體；三抗體= Fab-scFv融合蛋白多功能重組抗體衍生物(CreativeBiolabs)；Duobody平台(Genmab)；鎖鑰結構(dock and lock)平台；鈕扣(KIH)平台；人源化雙特異性IgG抗體(REGN1979) (Regeneron)；Mab2雙特異性抗體(F-Star)；DVD-lg =雙可變域免疫球蛋白(AbbVie)；κ-λ體；TBTI =四價雙特異性串聯Ig (tetravalent bispecific tandem Ig)；及CrossMab (Roche)、及上述中之任一者的表位結合片段。The antibodies and their fragments described herein include, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinant antibodies, multispecific antibodies (e.g., including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, intracellular antibodies, single-chain Fv (scFv) (e.g., including monospecific, bispecific, etc.), camelification antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fv (sdFv), anti-specific (anti-Id) antibodies, Fab-in-tandem IgG (FIT-IgG); DVD-IgG; heterozygous fusion tumors (quadroma or tetradoma); anti-carrier protein platform (Pieris); diabody; single-chain bichain antibody (single-chain... Diabody; tandem single-chain Fv fragment; TandAb, trispecific Ab (Affimed); Darts dual-affinity retargeting (Macrogenics); bispecific Xmab (Xencor); bispecific T cell conjugate (BiTE; Amgen; 55kDa); triple antibody; triple antibody = Fab-scFv fusion protein multifunctional recombinant antibody derivative (CreativeBiolabs); Duobody platform (Genmab); dock and lock platform; button (KIH) platform; humanized bispecific IgG antibody (REGN1979) (Regeneron); Mab2 bispecific antibody (F-Star); DVD-lg = bivariate immunoglobulin (AbbVie); κ-λ body; TBTI =Tetravalent bispecific tandem Ig; and CrossMab (Roche), and epitope binding fragments of any of the above.

典型的抗體分子包括重鏈可變區(VH)及輕鏈可變區(VL)。可變區係抗體分子之N端處胺基酸組成及排列變化較大的區。特異性結合的位點（亦即，抗原結合位點）用以判定抗體識別的特異性。VH及VL區可進一步細分成高度變異區，亦稱為「互補決定區(complementarity determining region)」(CDR)，其中散布稱為架構區(FR)之更具保守性的區。各VH及VL通常由三個CDR及四個FR組成，從胺基端到羧基端按以下順序排列：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。 例示性抗FAP抗體或其FAP抗原結合片段 Typical antibody molecules include a variable heavy chain region (VH) and a variable light chain region (VL). The variable region is the N-terminal region of the antibody molecule where the amino acid composition and arrangement vary considerably. The specific binding site (i.e., the antigen-binding site) is used to determine the specificity of antibody recognition. The VH and VL regions can be further subdivided into highly variable regions, also known as "complementarity determining regions" (CDRs), within which more conserved regions called structural regions (FRs) are scattered. Each VH and VL typically consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Example anti-FAP antibody or its FAP antigen-binding fragment

在一些實施例中，本揭露提供新型抗FAP抗體或其抗原結合片段的實例，該新型抗FAP抗體或其抗原結合片段包括重鏈互補決定區(CDR) HCDR1、HCDR2、及HCDR3、及輕鏈互補決定區LCDR1、LCDR2、及LCDR3。架構區及CDR之範圍可使用所屬技術領域中已知的方法精確識別，例如藉由Kabat定義、Chothia定義、AbM定義、IMGT定義、Honegger定義、及/或Contact定義，所有其等皆係所屬技術領域中熟知的。參見例如Kabat, E.A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242；Chothia et al., (1989) Nature 342:877；Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917；Al-lazikani et al. (1997) J. Molec. Biol. 273:927-948；IMGT ®, the international ImMunoGeneTics information system ®http://www.imgt.org, Lefranc, M.-P. et al., Nucleic Acids Res., 27:209-212 (1999)；Ruiz, M. et al., Nucleic Acids Res., 28:219-221 (2000)；Lefranc, M.-P., Nucleic Acids Res., 29:207-209 (2001)；Lefranc, M.-P., Nucleic Acids Res., 31:307-310 (2003)；Lefranc, M.-P. et al., In Silico Biol., 5, 0006 (2004) [Epub], 5:45-60 (2005)；Lefranc, M.-P. et al., Nucleic Acids Res., 33:D593-597 (2005)；Lefranc, M.-P. et al., Nucleic Acids Res., 37:D1006-1012 (2009)；Lefranc, M.-P. et al., Nucleic Acids Res., 43:D413-422 (2015)；Honegger and Plückthun, J. Mol. Biol. 309: 657-670 (2001)；及Almagro, J. Mol. Recognit.17:132‑143 (2004)。亦請參見hgmp.mrc.ac.uk and bioinf.org.uk/abs。 In some embodiments, this disclosure provides examples of novel anti-FAP antibodies or antigen-binding fragments thereof, which include heavy chain complementarity-determining regions (CDRs) HCDR1, HCDR2, and HCDR3, and light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3. The extent of the structural regions and CDRs can be precisely identified using methods known in the art, such as by Kabat, Chothia, AbM, IMGT, Honegger, and/or Contact definitions, all of which are well known in the art. See, for example, Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242; Chothia et al., (1989) Nature 342:877; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917; Al - lazikani et al. (1997) J. Molec. Biol. 273:927-948; M. et al., Nucleic Acids Res., 28:219-221 (2000); Lefranc, M.-P., Nucleic Acids Res., 29:207-209 (2001); Lefranc, M.-P., Nucleic Acids Res., 31:307-310 (2003); (2004) [Epub], 5:45-60 (2005); Lefranc, M.-P. et al., Nucleic Acids Res., 33:D593-597 (2005); Lefranc, M.-P. et al., Nucleic Acids Res., 37:D1006-1012 (2009); al., Nucleic Acids Res., 43:D413-422 (2015); Honegger and Plückthun, J. Mol. Biol . 309: 657-670 (2001); and Almagro, J. Mol. Recognit.17:132-143 (2004). See also hgmp.mrc.ac.uk and bioinf.org.uk/abs.

在一些實施例中，抗FAP抗體或其片段（包括人類抗FAP抗體或其片段）的CDR可根據任何適合的編號系統來判定。在一些實施例中，CDR由所屬技術領域中具有通常知識者熟知的Kabat系統定義。Kabat互補決定區(CDR)係基於序列變異性，且係最常用的（Kabat et al., (1971) Ann. NY Acad. Sci. 190:382-391及Kabat, et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242）。Chothia係指結構環的位置(Chothia and Lesk J. Mol. Biol. 196:901-917 (1987))。本文中所使用之編號系統係Kabat、IMGT、Honegger、及Chothia（參見例如 表 7）。 In some embodiments, the CDR of an anti-FAP antibody or fragment thereof (including human anti-FAP antibodies or fragment thereof) can be determined according to any suitable numbering system. In some embodiments, the CDR is defined by the Kabat system, which is well known to those of ordinary skill in the art. The Kabat complementarity-determining region (CDR) is based on sequence variability and is the most commonly used (Kabat et al., (1971) Ann. NY Acad. Sci. 190:382-391 and Kabat, et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242). Chothia refers to the location of the structural loop (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). The numbering systems used in this paper are Kabat, IMGT, Honegger, and Chothia (see Table 7 for example).

在一些實施例中，LCDR1包括如KTNQNVDYX 1GNTFMH (SEQ ID NO: 23)所示之胺基酸序列，其中X 1係N或S；LCDR2包括如LASNLAS (SEQ ID NO: 24)所示之胺基酸序列，LCDR3包括如QQSRNLPYT (SEQ ID NO: 25)所示之胺基酸序列；HCDR1包括如IYGVN (SEQ ID NO: 26)所示之胺基酸序列，HCDR2包括如AIWSGGRKDYX 2LSLKS (SEQ ID NO: 27)所示之胺基酸序列，其中X 2係N或S，HCDR3包括如SQDMPGYFDY (SEQ ID NO: 28)所示之胺基酸序列。在一些實施例中，LCDR1包括如SASSRVGYMH (SEQ ID NO: 29)所示之胺基酸序列，LCDR2包括如DTSKLAS (SEQ ID NO: 30)所示之胺基酸序列，LCDR3包括如FQGSGYPFT (SEQ ID NO: 31)所示之胺基酸序列；HCDR1包括如TAGMSVG (SEQ ID NO: 32)所示之胺基酸序列，HCDR2包括如DIWWDDKKHYNPSLKD (SEQ ID NO: 33)所示之胺基酸序列，HCDR3包括如DMIFNFYFDV (SEQ ID NO: 34)所示之胺基酸序列。 In some embodiments, LCDR1 includes an amino acid sequence as shown in KTNQNVDYX 1 GNTFMH (SEQ ID NO: 23), wherein X1 is N or S; LCDR2 includes an amino acid sequence as shown in LASNLAS (SEQ ID NO: 24); LCDR3 includes an amino acid sequence as shown in QQSRNLPYT (SEQ ID NO: 25); HCDR1 includes an amino acid sequence as shown in IYGVN (SEQ ID NO: 26); HCDR2 includes an amino acid sequence as shown in AIWSGGRKDYX 2 LSLKS (SEQ ID NO: 27), wherein X2 is N or S; and HCDR3 includes an amino acid sequence as shown in SQDMPGYFDY (SEQ ID NO: 28). In some embodiments, LCDR1 includes an amino acid sequence as shown in SASSRVGYMH (SEQ ID NO: 29), LCDR2 includes an amino acid sequence as shown in DTSKLAS (SEQ ID NO: 30), and LCDR3 includes an amino acid sequence as shown in FQGSGYPFT (SEQ ID NO: 31); HCDR1 includes an amino acid sequence as shown in TAGMSVG (SEQ ID NO: 32), HCDR2 includes an amino acid sequence as shown in DIWWDDKKHYNPSLKD (SEQ ID NO: 33), and HCDR3 includes an amino acid sequence as shown in DMIFNFYFDV (SEQ ID NO: 34).

在一些實施例中，抗FAP抗體或其抗原結合片段包括重鏈可變區(VH)及輕鏈(VL)，其中VH及/或VL包括上文所描述之CDR。在一些實施例中，抗FAP抗體或其抗原結合片段包括與如SEQ ID NO: 35所示之胺基酸序列具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、100%同一性的VH，及與如SEQ ID NO: 36所示之胺基酸序列具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、100%同一性的VL。在一些實施例中，抗FAP抗體或其抗原結合片段包括與如SEQ ID NO: 37所示之胺基酸序列具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、100%同一性的VH，及與如SEQ ID NO: 38所示之胺基酸序列具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、100%同一性的VL。In some embodiments, the anti-FAP antibody or its antigen-binding fragment includes a heavy chain variable region (VH) and a light chain (VL), wherein the VH and/or VL include the CDR described above. In some embodiments, the anti-FAP antibody or its antigen-binding fragment includes a VH having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 35, and a VL having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 36. In some embodiments, the anti-FAP antibody or its antigen-binding fragment includes a VH having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 37, and a VL having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 38.

在一些實施例中，抗FAP抗體或其抗原結合片段包括衍生自免疫球蛋白之可結晶片段(Fc)區。在一些實施例中，Fc片段係衍生自IgG1、IgG2、IgG3、或IgG4。在一些實施例中，Fc片段係衍生自IgG4。在一些實施例中，與野生型IgG1、IgG2、IgG3、或IgG4相比，Fc片段包括一或多個胺基酸取代。在一些實施例中，Fc片段包括突變S228P。在一些實施例中，Fc片段包括LALAPG突變，亦即，L234A、L235A、及/或P329G。在一些實施例中，Fc片段係異二聚體的。In some embodiments, the anti-FAP antibody or its antigen-binding fragment includes a crystallizable fragment (Fc) region derived from an immunoglobulin. In some embodiments, the Fc fragment is derived from IgG1, IgG2, IgG3, or IgG4. In some embodiments, the Fc fragment is derived from IgG4. In some embodiments, the Fc fragment includes one or more amino acid substitutions compared to wild-type IgG1, IgG2, IgG3, or IgG4. In some embodiments, the Fc fragment includes the S228P mutation. In some embodiments, the Fc fragment includes the LALAPG mutation, i.e., L234A, L235A, and/or P329G. In some embodiments, the Fc fragment is a heterodimer.

在一些實施例中，抗FAP抗體或其抗原結合片段包含：重鏈(HC)區，其包含與如SEQ ID NO: 1、4、5、6、8、9、10、11、12、13、14、15、及16所示之胺基酸序列中之任一者包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；及/或輕鏈(LC)區，其包含與如SEQ ID NO: 2、3、及7所示之胺基酸序列中之任一者包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列。In some embodiments, the anti-FAP antibody or its antigen-binding fragment comprises: a heavy chain (HC) region containing an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the amino acid sequences shown in SEQ ID NO: 1, 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, and 16; and/or a light chain (LC) region containing an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the amino acid sequences shown in SEQ ID NO: 2, 3, and 7.

在一些實施例中，抗FAP抗體或其抗原結合片段包含HC1區，該區包含與如SEQ ID NO: 1、4、5、8、10、13、14、及15所示之胺基酸序列中之任一者包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列。In some embodiments, the anti-FAP antibody or its antigen-binding fragment includes an HC1 region containing an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with or identical to any of the amino acid sequences shown in SEQ ID NO: 1, 4, 5, 8, 10, 13, 14, and 15.

在一些實施例中，抗FAP抗體或其抗原結合片段包含HC1區，該區包含與如SEQ ID NO: 5、8、10、13、14、及15所示之胺基酸序列中之任一者包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列。In some embodiments, the anti-FAP antibody or its antigen-binding fragment includes an HC1 region containing an amino acid sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with or is identical to any of the amino acid sequences shown in SEQ ID NO: 5, 8, 10, 13, 14, and 15.

在一些實施例中，抗FAP抗體或其抗原結合片段包含HC2區，該區包含與如SEQ ID NO: 1、4、6、9、11、12、13、及16所示之胺基酸序列中之任一者包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列。In some embodiments, the anti-FAP antibody or its antigen-binding fragment includes an HC2 region containing an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with or identical to any of the amino acid sequences shown in SEQ ID NO: 1, 4, 6, 9, 11, 12, 13, and 16.

在一些實施例中，抗FAP抗體或其抗原結合片段包含HC2區，該區包含與如SEQ ID NO: 6、9、11、12、13、及16所示之胺基酸序列中之任一者包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列。In some embodiments, the anti-FAP antibody or its antigen-binding fragment includes an HC2 region containing an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with or identical to any of the amino acid sequences shown in SEQ ID NO: 6, 9, 11, 12, 13, and 16.

在一些實施例中，抗FAP抗體或其抗原結合片段包括具有如SEQ ID NO: 1所示之胺基酸序列的重鏈、及具有如SEQ ID NO: 2所示之胺基酸序列的輕鏈。在一些實施例中，抗FAP抗體或其抗原結合片段包括具有如SEQ ID NO: 3所示之胺基酸序列的重鏈、及具有如SEQ ID NO: 4所示之胺基酸序列的輕鏈。在一些實施例中，抗FAP抗體或其抗原結合片段包括具有SEQ ID NO: 4所述之胺基酸序列的重鏈、及具有SEQ ID NO: 3所述之胺基酸序列的輕鏈。抗FAP抗體的另外CDR、VH、VL、HC、及/或LC在本文中的FAP結合劑部分中詳細描述。In some embodiments, the anti-FAP antibody or its antigen-binding fragment comprises a heavy chain having the amino acid sequence shown in SEQ ID NO: 1 and a light chain having the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the anti-FAP antibody or its antigen-binding fragment comprises a heavy chain having the amino acid sequence shown in SEQ ID NO: 3 and a light chain having the amino acid sequence shown in SEQ ID NO: 4. In some embodiments, the anti-FAP antibody or its antigen-binding fragment comprises a heavy chain having the amino acid sequence described in SEQ ID NO: 4 and a light chain having the amino acid sequence described in SEQ ID NO: 3. Further CDR, VH, VL, HC, and/or LC of the anti-FAP antibody are described in detail in the FAP binding agent section herein.

在一些實施例中，抗FAP抗體或其抗原結合片段與人類、食蟹獼猴、及小鼠FAP交叉反應。In some embodiments, anti-FAP antibodies or their antigen-binding fragments cross-react with human, cynomolgus monkey, and mouse FAP.

經單離抗FAP抗體或其FAP抗原結合片段係單株抗體、人源化抗體、人類抗體、嵌合抗體、Fab、Fab’、F(ab’)2、Fv、scFv、(scFv)2、單鏈抗體分子、雙可變區抗體、單可變區抗體、線性抗體、V區、或由抗體片段形成之多特異性抗體。 人源化抗體 FAP antibodies or their FAP antigen-binding fragments can be monoclonal antibodies, humanized antibodies, human antibodies, chimeric antibodies, Fab, Fab’, F(ab’)2, Fv, scFv, (scFv)2, single-chain antibody molecules, bivariate antibodies, monovariate antibodies, linear antibodies, V-region antibodies, or multispecific antibodies formed from antibody fragments. Humanized antibodies

在一些實施例中，本文所提供之抗體係人源化抗體。在一些實施例中，嵌合抗體係人源化抗體。一般而言，非人類抗體經人源化以減少對人類之免疫性，同時保有親本非人類抗體之特異性及親和力。通常，人源化抗體包含一或多個可變域，其中CDR（或其部分）係衍生自非人類抗體，且FR（或其部分）係衍生自人類抗體序列。人源化抗體可選地亦將包含人類恆定區之至少一部分。在一些實施例中，人源化抗體中之一些FR殘基經來自非人類抗體（例如CDR殘基來源的抗體）之對應殘基取代，以例如回復或改善抗體特異性或親和力。In some embodiments, the antibodies provided herein are humanized antibodies. In some embodiments, chimeric antibodies are humanized antibodies. Generally, non-human antibodies are humanized to reduce immunity to humans while retaining the specificity and affinity of the parent non-human antibody. Typically, humanized antibodies contain one or more variable domains, wherein the CDR (or a portion thereof) is derived from the non-human antibody, and the FR (or a portion thereof) is derived from the human antibody sequence. Humanized antibodies may also optionally contain at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., an antibody from which the CDR residue is derived) to, for example, restore or improve antibody specificity or affinity.

人源化抗體及其製造方法係綜述於例如Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)，且進一步描述於例如Riechmann et al., Nature 332:323-329 (1988)；Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989)；美國專利第5,821,337、7,527,791、6,982,321、及7,087,409號；Kashmiri et al., Methods 36:25-34 (2005)（描述特異性判定區(specificity determining region, SDR)移植）；Padlan, Mol. Immunol. 28:489-498 (1991)（描述「重表面加工(resurfacing)」）；Dall'Acqua et al., Methods 36:43-60 (2005)（描述「FR改組(FR shuffling)」）；及Osbourn et al., Methods 36:61-68 (2005)及Klimka et al., Br. J. Cancer, 83:252-260 (2000)（描述「導向選擇(guided selection)」法用以FR改組）。 Humanized antibodies and their manufacturing methods are summarized in, for example, Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described in, for example, Riechmann et al ., Nature 332:323-329 (1988); Queen et al ., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); US Patents 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al ., Methods 36:25-34 (2005) (describing the transplantation of the specificity determining region (SDR); Padlan, Mol. Immunol. 28:489-498. (1991) (describes "resurfacing");Dall'Acqua et al ., Methods 36:43-60 (2005) (describes "FR shuffling"); and Osbourn et al ., Methods 36:61-68 (2005) and Klimka et al ., Br. J. Cancer, 83:252-260 (2000) (describes "guided selection" for FR shuffling).

可用於人源化之人類架構區包括但不限於：使用「最佳擬合(best-fit)」法所選擇之架構區（參見例如Sims et al. J. Immunol. 151:2296 (1993)）；衍生自輕鏈或重鏈可變區之特定子群的人類抗體之共有序列的架構區（參見例如Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992)；及Presta et al. J. Immunol., 151:2623 (1993)）；人類成熟（體細胞突變）架構區或人類種系架構區（參見例如Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)）；及衍生自篩選FR庫之架構區（參見例如Baca et al., J. Biol. Chem. 272:10678-10684 (1997)及Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)）。 人類抗體 Human skeletal regions that can be used for humanization include, but are not limited to: skeletal regions selected using the "best-fit" method (see, for example, Sims et al . J. Immunol. 151:2296 (1993)); skeletal regions containing the common sequences of human antibodies derived from specific subgroups of variable regions of the light or heavy chains (see, for example, Carter et al . Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al . J. Immunol., 151:2623 (1993)); human mature (somatic mutation) skeletal regions or human germline skeletal regions (see, for example, Almagro and Fransson, Front. Biosci. 13:1619-1633). (2008)); and structural regions derived from the screened FR library (see, for example, Baca et al ., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al ., J. Biol. Chem. 271:22611-22618 (1996)). Human antibodies

在一些實施例中，本文所提供之抗體係人類抗體。人類抗體可使用所屬技術領域中已知之各種技術產生。人類抗體係大致描述於van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001)及Lonberg, Curr. Opin. Immunol. 20:450-459 (2008)。In some embodiments, the antibody system provided herein is a human antibody. Human antibodies can be produced using various techniques known in the relevant art field. Human antibody systems are broadly described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

人類抗體可藉由投予免疫原至已經修飾以回應於抗原挑戰而產生完整人類抗體或具有人類可變區之完整抗體的基因轉殖動物來製備。此類動物一般含有全部或部分的人類免疫球蛋白基因座，其置換了內源免疫球蛋白基因座，或其係存在於染色體外或隨機整合至動物之染色體中。在此類基因轉殖小鼠中，通常已將內源免疫球蛋白基因座去活化。關於用於自基因轉殖動物獲得人類抗體之方法的綜述，參見Lonberg, Nat. Biotech. 23:1117-1125 (2005)。亦參見例如美國專利第6,075,181及6,150,584號（描述XENOMOUSE ™技術）；美國專利第5,770,429號（描述HUMAB ®技術）；美國專利第7,041,870號（描述K-M MOUSE ®技術）、及美國專利申請公開案第US 2007/0061900號（描述VELOCIMOUSE ®技術）。來自由此類動物所產生之完整抗體的人類可變區可經進一步修飾，例如藉由與不同的人類恆定區組合。 Human antibodies can be prepared by administering an immunogen to genetically modified animals that produce complete human antibodies in response to antigenic challenges or complete antibodies with human variable regions. These animals typically contain all or part of the human immunoglobulin loci, which replace endogenous immunoglobulin loci, or are located extrachromosomally or randomly integrated into the animal's chromosomes. In these genetically modified mice, the endogenous immunoglobulin loci are usually deactivated. For a review of methods for obtaining human antibodies from genetically modified animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, for example, U.S. Patents 6,075,181 and 6,150,584 (describing XENOMOUSE ™ technology); U.S. Patent 5,770,429 (describing HUMAB® technology); U.S. Patent 7,041,870 (describing KM MOUSE® technology); and U.S. Patent Application Publication No. US 2007/0061900 (describing VELOCIMOUSE® technology). The human variable region of the intact antibody produced by such animals can be further modified, for example, by combining it with different human constant regions.

人類抗體亦可藉由基於融合瘤之方法製造。已描述用於產生人類單株抗體之人類骨髓瘤及小鼠-人類異源骨髓瘤細胞系。（參見例如Kozbor J. Immunol., 133: 3001 (1984)；Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (1987)；及Boerner et al., J. Immunol., 147: 86 (1991)。）經由人類B細胞融合瘤技術所產生之人類抗體亦描述於Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006)中。額外的方法包括例如在美國專利第7,189,826號（描述從融合瘤細胞系產生單株人類IgM抗體）及Ni, Xiandai Mianyixue, 26(4):265-268 (2006)（描述人類-人類融合瘤）中所述者。人類融合瘤技術（Trioma技術）亦描述於Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005)及Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3): 185-91 (2005)。 Human antibodies can also be produced using fusion tumor-based methods. Human myeloma and mouse-human heterologous myeloma cell lines used to produce human monoclonal antibodies have been described. (See, for example, Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al ., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (1987); and Boerner et al ., J. Immunol., 147: 86 (1991).) Human antibodies produced via human B-cell fusion tumor technology are also described in Li et al ., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additional methods include, for example, those described in U.S. Patent No. 7,189,826 (description of monoclonal human IgM antibodies from fusion tumor cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (description of human-human fusion tumors). The human fusion tumor technique (Trioma technique) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3): 185-91 (2005).

人類抗體亦可藉由單離選自人源噬菌體展示庫之可變域序列來產生。然後可將此類可變域序列與所欲之人類恆定域組合。用於從抗體庫選出人類抗體之技術係描述於下。Human antibodies can also be generated by singling variable domain sequences selected from a human phage display library. These variable domain sequences can then be combined with desired human constant domains. The techniques for selecting human antibodies from an antibody library are described below.

抗體可藉由針對具有所欲一或多種活性之抗體來篩檢組合庫而單離。例如，關於產生噬菌體展示庫及針對擁有所欲結合特徵之抗體來篩檢此類庫，各種方法係所屬技術領域中已知的。此類方法係綜述於例如Hoogenboom et al. in Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001)中且進一步描述於例如McCafferty et al, Nature 348:552-554 (1990)；Clackson et al, Nature 352: 624-628 (1991)；Marks et al, J. Mol. Biol 222: 581-597(1992)；Marks and Bradbury, in Methods in Molecular Biology 248: 161-175 (Lo, ed., Human Press, Totowa, NJ, 2003)；Sidhu et al, J. Mol. Biol. 338(2) (2004): 299-310；Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004)；Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004)；及Lee et al, (2004) J. Immunol. Methods 284(1-2): 119-132、及PCT申請案WO 99/10494。 Antibodies can be isolated by screening a library of combinations for antibodies that have one or more desired activities. For example, various methods for generating phage display libraries and screening such libraries for antibodies that have desired binding characteristics are known in the relevant technical field. Such methods are summarized in, for example, Hoogenboom et al . in Methods in Molecular Biology 178: 1-37 (O'Brien et al ., ed., Human Press, Totowa, NJ, 2001) and further described in, for example, McCafferty et al ., Nature 348:552-554 (1990); Clackson et al. , Nature 352: 624-628 (1991); Marks et al ., J. Mol. Biol 222: 581-597 (1992); Marks and Bradbury, in Methods in Molecular Biology 248: 161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al ., J. Mol. Biol. 338(2) (2004): 299-310; Lee et al ., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al ., (2004) J. Immunol. Methods 284(1-2): 119-132, and PCT application WO 99/10494.

在某些噬菌體展示方法中，VH及VL基因庫係分開地藉由聚合酶鏈反應(polymerase chain reaction, PCR)來選殖並隨機重組於噬菌體庫中，然後可針對抗原結合噬菌體進行篩檢，如描述於Winter et al., Ann. Rev. Immunol., 12:433-455 (1994)。噬菌體一般展示抗體片段，以單鏈Fv (scFv)片段或以Fab片段。來自固定化來源之庫提供對免疫原具有高親和力之抗體而無需構築融合瘤。替代地，可對天然庫（例如，來自人類）進行選殖，以對廣泛範圍的非自體抗原及自體抗原提供單一抗體來源而無需任何免疫化，如由Griffiths et al., EMBO J 12:725-734 (1993)所述。最後，天然庫亦可藉由對來自幹細胞之未經重排V基因段進行選殖，並使用含有隨機序列之PCR引子編碼高度可變CDR3區且在體外達成重排來製造，如由Hoogenboom and Winter, J. Mol. Biol, 227:381-388 (1992)所述。描述人類抗體噬菌體庫之專利公開案包括例如：美國專利第5,750,373號、及美國專利公開案第2005/0079574、2005/0119455、2005/0266000、2007/0117126、2007/0160598、2007/0237764、2007/0292936、及2009/0002360號。 In some phage display methods, VH and VL gene libraries are separately selected and randomly recombined into the phage library via polymerase chain reaction (PCR), allowing for screening of antigen-binding phages, as described in Winter et al ., Ann. Rev. Immunol., 12:433-455 (1994). Phages typically display antibody fragments, either as single-stranded Fv (scFv) fragments or as Fab fragments. Libraries derived from immobilized sources provide antibodies with high affinity for immunogens without the need to construct fusion tumors. Alternatively, natural libraries (e.g., from humans) can be selected to provide a single source of antibodies against a wide range of non-autoantigens and autoantigens without any immunization, as described by Griffiths et al ., EMBO J 12:725-734 (1993). Finally, natural libraries can also be produced by selecting unrearranged V gene segments from stem cells and using PCR primers containing random sequences to encode highly variable CDR3 regions and achieve rearrangement in vitro, as described by Hoogenboom and Winter, J. Mol. Biol, 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example, U.S. Patent No. 5,750,373, and U.S. Patent Publications Nos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936, and 2009/0002360.

在一些實施例中，提供一種嵌合人類抗FAP抗體，其中該抗體包含來自結合FAP之人類抗體的可變區及來自不同人類抗體的恆定區。在一些實施例中，提供一種嵌合人類抗FAP抗體，其中該抗體包含來自結合FAP之人類抗體的CDR及來自不同人類抗體的架構。在一些實施例中，抗體係非天然存在人類抗體。In some embodiments, a chimeric human anti-FAP antibody is provided, wherein the antibody includes a variable region derived from a human antibody bound to FAP and a constant region derived from a different human antibody. In some embodiments, a chimeric human anti-FAP antibody is provided, wherein the antibody includes a CDR derived from a human antibody bound to FAP and a structure derived from a different human antibody. In some embodiments, the antibody is a non-naturally occurring human antibody.

在一些實施例中，人類抗FAP抗體包含一或多個人類恆定區。在一些實施例中，人類重鏈恆定區具有選自IgA、IgG、IgD、及IgE之同型。在一些實施例中，人類輕鏈恆定區具有選自κ及λ之同型。在一些實施例中，本文所述之人類抗體包含人類IgG恆定區。在一些實施例中，本文所述之人類抗體包含人類IgG4重鏈恆定區。在一些實施例中，本文所述之人類抗體包含人類IgG4恆定區及人類κ輕鏈。In some embodiments, the human anti-FAP antibody comprises one or more human constant regions. In some embodiments, the human heavy chain constant region has isotypes selected from IgA, IgG, IgD, and IgE. In some embodiments, the human light chain constant region has isotypes selected from κ and λ. In some embodiments, the human antibody described herein comprises a human IgG constant region. In some embodiments, the human antibody described herein comprises a human IgG4 heavy chain constant region. In some embodiments, the human antibody described herein comprises a human IgG4 constant region and a human κ light chain.

在一些實施例中，當效應功能係所欲的時，則選擇包含人類IgG1重鏈恆定區或人類IgG3重鏈恆定區之人類抗FAP抗體。在一些實施例中，當效應功能係非所欲的時，則選擇包含人類IgG4或IgG2重鏈恆定區之人類抗FAP抗體。In some embodiments, when the desired effect is achieved, a human anti-FAP antibody containing the human IgG1 heavy chain constant region or the human IgG3 heavy chain constant region is selected. In some embodiments, when the desired effect is not achieved, a human anti-FAP antibody containing the human IgG4 or IgG2 heavy chain constant region is selected.

當描述人類抗體時，參考抗體構築體的可能序列的屬，而非抗體的來源。 多特異性抗體 When describing human antibodies, refer to the genus of the possible sequence of the antibody building blocks, rather than the origin of the antibody. Multispecific antibodies

在一些實施例中，本文所提供之抗體係多特異性抗體，例如雙特異性抗體。多特異性抗體係對至少兩個不同位點具有結合特異性的單株抗體，例如不同抗原上的不同表位或同一抗原上的不同表位。在某些實施例中，多特異性抗體具有三或更多種結合特異性。在某些實施例中，結合特異性中的一者係針對FAP，且另一特異性係針對任何其他抗原。在某些實施例中，雙特異性抗體可結合至FAP的兩個（或更多個）不同表位。多特異性（例如雙特異性）抗體亦可用以將細胞毒性劑或細胞定位至表現FAP之細胞。多特異性抗體可製備為全長抗體或抗體片段。In some embodiments, the antibodies provided herein are multispecific antibodies, such as bispecific antibodies. Multispecific antibodies are monoclonal antibodies that have binding specificity to at least two different sites, such as different epitopes on different antigens or different epitopes on the same antigen. In some embodiments, multispecific antibodies have three or more binding specificities. In some embodiments, one binding specificity is against FAP, and another specificity is against any other antigen. In some embodiments, bispecific antibodies can bind to two (or more) different epitopes of FAP. Multispecific (e.g., bispecific) antibodies can also be used to target cytotoxic agents or cells to cells expressing FAP. Multispecific antibodies can be prepared as full-length antibodies or antibody fragments.

用於製備多特異性抗體的技術包括但不限於具有不同特異性的兩個免疫球蛋白重鏈-輕鏈對的重組共表現（參見Milstein and Cuello, Nature 305: 537 (1983)）及「鈕扣」工程（參見例如美國專利第5,731,168號及Atwell et al., J. Mol. Biol. 270:26 (1997)）。多特異性抗體亦可藉由下列來製備：工程改造靜電操縱效應來製備抗體Fc-異二聚體分子（參見例如WO 2009/089004）；交聯二或更多個抗體或片段（參見例如美國專利第4,676,980號及Brennan et al., Science, 229: 81 (1985)）；使用白胺酸拉鍊產生雙特異性抗體（參見例如Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)及WO 2011/034605）；使用普通輕鏈技術來規避輕鏈錯配問題（參見例如WO 98/50431）；使用「雙鏈抗體(diabody)」技術製造雙特異性抗體片段（參見例如Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)）；及使用單鏈Fv (sFv)二聚體（參見例如Gruber et al., J. Immunol., 152:5368 (1994)）；及製備三特異性抗體，如例如Tutt et al. J. Immunol. 147: 60 (1991)中所述。 Techniques for preparing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein and Cuello, Nature 305: 537 (1983)) and "button" engineering (see, for example, U.S. Patent No. 5,731,168 and Atwell et al ., J. Mol. Biol. 270:26 (1997)). Multispecific antibodies can also be prepared by: engineering electrostatic manipulation to prepare antibody Fc-heterodimer molecules (see, for example, WO 2009/089004); crosslinking two or more antibodies or fragments (see, for example, US Patent No. 4,676,980 and Brennan et al ., Science, 229: 81 (1985)); using leucine zippers to produce bispecific antibodies (see, for example, Kostelny et al ., J. Immunol., 148(5):1547-1553 (1992) and WO 2011/034605); and using conventional light chain technology to avoid light chain mismatch problems (see, for example, WO 98/50431); the fabrication of bispecific antibody fragments using "diabody" technology (see, for example, Hollinger et al ., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and the fabrication of trispecific antibodies using single-chain Fv (sFv) dimers (see, for example, Gruber et al ., J. Immunol., 152:5368 (1994)); as described, for example, Tutt et al . J. Immunol. 147: 60 (1991).

本文所提供之抗FAP抗體可與第二分子融合或接合。本文所提供之多特異性抗FAP抗體可與額外分子融合或接合。本文進一步描述融合蛋白及抗體接合物。 雙特異性抗體或其抗原結合片段 The anti-FAP antibodies presented in this article can fuse or bind to a second molecule. The multispecific anti-FAP antibodies presented in this article can fuse or bind to additional molecules. This article further describes fusion proteins and antibody conjugates. Bispecific antibodies or their antigen-binding fragments

本文提供一種雙特異性抗體、其抗原結合片段、或其用途。在一些實施例中，雙特異性抗體包含第一結合部份及第二結合部份。在一些實施例中，第一結合部份包含本文所述之FAP抗原結合部份。在一些實施例中，抗原結合部份包括直接與抗原交互作用並與其結合的抗體的特定區或組分。該部份可包括重鏈及/或輕鏈的一或多個可變區，該一或多個可變區形成抗原結合位點。在一些實施例中，FAP抗原結合部份包括本文所述之抗FAP抗體或其抗原結合片段的部分或全長。FAP抗原結合部份包含本文所述之CDR、VH、VL、HC（包括HC1或HC2）、及/或LC中之任一或多者。This document provides a bispecific antibody, its antigen-binding fragment, or its use. In some embodiments, the bispecific antibody includes a first binding moiety and a second binding moiety. In some embodiments, the first binding moiety includes the FAP antigen-binding moiety described herein. In some embodiments, the antigen-binding moiety includes a specific region or component of the antibody that directly interacts with and binds to an antigen. The moiety may include one or more variable regions of a heavy chain and/or light chain that form antigen-binding sites. In some embodiments, the FAP antigen-binding moiety includes a portion or the full length of an anti-FAP antibody or its antigen-binding fragment described herein. The FAP antigen-binding moiety includes any or more of CDR, VH, VL, HC (including HC1 or HC2), and/or LC described herein.

在一些實施例中，本文所述之雙特異性抗體包含兩條重鏈，其中各重鏈結合不同的表位。在一些實施例中，本文所述之雙特異性抗體包含兩條重鏈，其中各重鏈結合相同的表位。各重鏈可在一端具有可變域(VH)，隨後係數個恆定域（三個或四個恆定域，CH1、CH2、CH3、及CH4，取決於抗體類別）。在一些實施例中，本文所述之雙特異性抗體包含一或多條輕鏈。各輕鏈可在一端具有可變域(VL)且在其另一端具有恆定域(CL)；輕鏈的恆定域與重鏈的第一恆定域(CH1)對準，且輕鏈可變域與重鏈的可變域對準。在一些實施例中，輕鏈包含κ輕鏈或λ輕鏈。雙特異性抗體（諸如κ或λ抗體）可使用多種所屬技術領域認可的技術中之任一者來製備，包括WO 2012/023053中所揭示者，其內容特此以引用方式全部併入本文中。In some embodiments, the bispecific antibody described herein comprises two heavy chains, each binding to a different epitope. In some embodiments, the bispecific antibody described herein comprises two heavy chains, each binding to the same epitope. Each heavy chain may have a variable domain (VH) at one end, followed by a number of constant domains (three or four constant domains, CH1, CH2, CH3, and CH4, depending on the antibody class). In some embodiments, the bispecific antibody described herein comprises one or more light chains. Each light chain may have a variable domain (VL) at one end and a constant domain (CL) at the other end; the constant domain of the light chain aligns with the first constant domain (CH1) of the heavy chain, and the variable domain of the light chain aligns with the variable domain of the heavy chain. In some embodiments, the light chain comprises a κ light chain or a λ light chain. Bispecific antibodies (such as κ or λ antibodies) may be prepared using any of a variety of techniques recognized in the respective fields of art, including those disclosed in WO 2012/023053, the entire contents of which are hereby incorporated by reference.

在一些實施例中，具有所欲結合特異性的抗體可變域可連接至免疫球蛋白恆定域序列以形成雙特異性抗體。在一些實施例中，與免疫球蛋白重鏈恆定域融合，該免疫球蛋白重鏈恆定域包含鉸鏈區、CH2區、及CH3區的至少一部分。在一些實施例中，含有輕鏈結合所需位點的第一重鏈恆定區(CH1)存在於融合體中的至少一者中。編碼免疫球蛋白重鏈融合體及（若為所欲）免疫球蛋白輕鏈的DNA可插入到單獨的表現載體中，且可共轉染到適合的宿主生物體中。In some embodiments, an antibody variable domain having the desired binding specificity may be linked to an immunoglobulin constant domain sequence to form a bispecific antibody. In some embodiments, fusion with an immunoglobulin heavy chain constant domain comprising at least a portion of a hinge region, a CH2 region, and a CH3 region is present. In some embodiments, a first heavy chain constant region (CH1) containing the site required for light chain binding is present in at least one of the fusion bodies. The DNA encoding the immunoglobulin heavy chain fusion body and (if desired) the immunoglobulin light chain may be inserted into a separate expression vector and may be co-transfected into a suitable host organism.

在一些實施例中，本文構築體中的一對抗體分子之間的界面經工程改造以使從重組細胞培養物中回復的異二聚體的百分比最大化。在該方法中，來自第一抗體分子界面的一或多個小胺基酸側鏈經較大的側鏈置換以形成突起或鈕（例如酪胺酸或色胺酸）。藉由用較小的胺基酸側鏈取代大的胺基酸側鏈，在第二抗體分子的界面上建立與（多條）大側鏈相同或相似尺寸的補償性腔或孔（例如絲胺酸、蘇胺酸、纈胺酸、或丙胺酸）。這提供了一種相對於其他非所要的終產物（諸如同二聚體）用於增加異二聚體產率的機制。In some embodiments, the interface between a pair of antibody molecules in this construct is engineered to maximize the percentage of heterodimers recovered from recombinant cell cultures. In this approach, one or more small amino acid side chains from the interface of the first antibody molecule are replaced by larger side chains to form protrusions or buttons (e.g., tyrosine or tryptophan). By replacing the large amino acid side chains with smaller ones, compensating cavities or pores (e.g., serine, threonine, volamine, or alanine) of the same or similar size as the (multiple) large side chains are created at the interface of the second antibody molecule. This provides a mechanism for increasing heterodimer yield relative to other undesirable end products (such as isomers).

文獻中已經描述了從抗體功能片段產生雙特異性抗體的技術。例如，可使用化學鍵聯來製備雙特異性抗體。雙特異性抗體可用作選擇性固定酶的試劑。Techniques for generating bispecific antibodies from antibody functional fragments have been described in the literature. For example, chemical bonding can be used to prepare bispecific antibodies. Bispecific antibodies can be used as reagents for selectively immobilizing enzymes.

亦描述了直接從重組細胞培養物中製備及單離雙特異性抗體功能片段的各種技術。例如，已經使用白胺酸拉鍊產生了雙特異性抗體。來自Fos及Jun蛋白的白胺酸拉鍊肽藉由基因融合與兩種不同抗體的Fab'部分連接。抗體同二聚體在鉸鏈區被還原形成單體，且接著重新氧化形成抗體異二聚體。該方法亦可用於抗體同二聚體的產生。「雙鏈抗體」技術為製備雙特異性抗體功能片段提供替代的機制。功能片段包含藉由連接子連接至輕鏈可變域(VL)的重鏈可變域(VH)，該連接子太短而不允許同一鏈上的兩個域之間配對。因此，一個功能片段的VH及VL域被強制與另一功能片段的互補VL及VH域配對，藉以形成兩個目標結合位點。製備雙特異性抗體功能片段的另一種策略包括使用單鏈Fv (sFv)二聚體。Various techniques for preparing and isolating bispecific antibody functional fragments directly from recombinant cell cultures are also described. For example, bispecific antibodies have been generated using leucine zippers. Leucine zipper peptides from Fos and Jun proteins are linked to the Fab' moieties of two different antibodies via gene fusion. The antibody homodimer is reduced to a monomer in the hinge region and then re-oxidized to form an antibody heterodimer. This method can also be used for the generation of antibody homodimers. The "bichain antibody" technique provides an alternative mechanism for preparing bispecific antibody functional fragments. The functional fragment includes a heavy chain variable domain (VH) linked to a light chain variable domain (VL) by a linker that is too short to allow pairing between the two domains on the same chain. Therefore, the VH and VL domains of one functional fragment are forced to pair with complementary VL and VH domains of another functional fragment to form two target binding sites. Another strategy for preparing bispecific antibody functional fragments involves using single-chain Fv (sFv) dimers.

考慮了具有多於兩價的抗體。例如，可製備三特異性抗體。例示性雙特異性抗體可結合兩個不同的表位，其中至少一個表位源自本文所述之目標。替代地，免疫球蛋白分子的目標臂可與結合至TACR（諸如本文所述之TACR）的臂組合。雙特異性抗體亦可用以將細胞毒性劑引導至表現特定蛋白質的細胞。此等抗體可具有目標結合臂及結合細胞毒性劑的臂，諸如本文所述者。Antibodies with more than two valents are considered. For example, trispecific antibodies can be prepared. An illustrative bispecific antibody may bind to two distinct epitopes, at least one of which originates from the target described herein. Alternatively, the target arm of an immunoglobulin molecule may be combined with an arm that binds to a TACR (such as the TACR described herein). Bispecific antibodies can also be used to direct cytotoxic agents to cells expressing specific proteins. Such antibodies may have a target-binding arm and an arm that binds to a cytotoxic agent, as described herein.

已使用若干策略來產生此類多特異性分子（例如雙特異性分子、三特異性分子），諸如抗體功能片段的化學交聯、強制異二聚化、四源雜交瘤技術、經由多肽連接子之抗體功能片段的融合、及單域抗體的使用。重組DNA技術的可用性導致了多種雙特異性抗體形式的產生。連接子及突變經常被引入抗體之不同區中，以強制異二聚體形成或將不同的結合部份連接成單一分子。Several strategies have been used to generate such multispecific molecules (e.g., bispecific and trispecific molecules), such as chemical crosslinking of antibody functional fragments, forced heterodimerization, tetralogous hybridization, fusion of antibody functional fragments via polypeptide linkers, and the use of single-domain antibodies. The availability of recombinant DNA technology has led to the generation of various forms of bispecific antibodies. Linkers and mutations are often introduced into different regions of the antibody to force heterodimerization or to link different binding sites into a single molecule.

在一些實施例中，雙特異性抗體或其抗原結合片段在FAP抗原結合部份之N端包括衍生自免疫球蛋白之Fc片段。在一些實施例中，Fc片段係衍生自IgG1、IgG2、IgG3、或IgG4，可選地，Fc片段係衍生自IgG4。在一些實施例中，Fc片段包括突變S228P。在一些實施例中，Fc片段包括選自由下列所組成之群組的一或多個修飾：鈕扣、DDKK、CH3之靜電轉向、DuoBody、SEEDbodies、cFAE、XmAb、Azymetric、及BEAT ®。在一些實施例中，Fc片段包括修飾鈕扣及/或DDKK。 In some embodiments, the bispecific antibody or its antigen-binding fragment includes an Fc fragment derived from an immunoglobulin at the N-terminus of the FAP antigen-binding region. In some embodiments, the Fc fragment is derived from IgG1, IgG2, IgG3, or IgG4, and optionally, the Fc fragment is derived from IgG4. In some embodiments, the Fc fragment includes the mutant S228P. In some embodiments, the Fc fragment includes one or more modifications selected from the group consisting of: button, DDKK, electrostatic switching of CH3, DuoBody, SEEDbodies, cFAE, XmAb, Azymetric, and BEAT® . In some embodiments, the Fc fragment includes modified button and/or DDKK.

鈕扣突變可藉由在CH3域中引入突變來改變接觸界面，從而強制兩條不同的IgG重鏈配對。在一條鏈上引入具有大側鏈的胺基酸，以形成「鈕」。相反，大的胺基酸經具有短側鏈的胺基酸置換，從而在另一CH3域中建立「孔」。藉由共表現此等兩條重鏈，可觀察到相對於同二聚體形成（「孔-孔」或「鈕-鈕」）之大於90%的異二聚體形成（「鈕-孔」）。基於人類IgG及人類IgA序列的經工程改造之股交換工程改造域(strand- exchange engineered domain, SEED)人類CH3域亦可導致形成可攜帶兩種不同特異性的異二聚體分子。最近對「鈕扣」方法進行改善；「CrossMab」已描述於WO 2009/080253 Al。除了「鈕扣」突變之外，該方法亦涉及輕鏈及重鏈域中之一些者的交換。DDKK係介導靜電轉向效應的修飾，其用以增強抗體Fc異二聚體形成，特別如Gunasekaran et al., (J. Biol. Chem. 2010,19637-19646)所述。因此，在一些實施例中，本文提供一或多個異二聚化修飾。Button mutations can alter the contact interface by introducing a mutation into the CH3 domain, thereby forcing the pairing of two distinct IgG heavy chains. An amino acid with a large sidechain is introduced into one chain to form a "button." Conversely, a large amino acid is replaced by an amino acid with a short sidechain, thus creating a "pore" in the other CH3 domain. By co-expressing these two heavy chains, greater than 90% heterodimer formation ("button-pore") can be observed relative to homodimer formation ("pore-pore" or "button-button"). Engineered strand-exchange-engineered domains (SEED) of the human CH3 domain, based on human IgG and human IgA sequences, can also lead to the formation of heterodimer molecules capable of carrying two different specificities. Recent improvements have been made to the "button" method; "CrossMab" has been described in WO 2009/080253 A1. In addition to the "button" mutation, this method also involves the exchange of some components in the light and heavy chain domains. DDKK is a modification that mediates electrostatic switching effects to enhance the formation of antibody Fc heterodimers, particularly as described by Gunasekaran et al., (J. Biol. Chem. 2010, 19637-19646). Therefore, in some embodiments, this paper provides one or more heterodimerization modifications.

在一些實施例中，第二結合部份結合及/或活化第二目標。在一些實施例中，第二目標係腫瘤相關細胞受體(tumor associated cell receptor, TACR)。TACR可在腫瘤細胞上或在與腫瘤細胞或腫瘤微環境相關聯之免疫細胞上表現。在一些實施例中，第二結合部份結合TACR。在一些實施例中，第二結合部份活化TACR。在一些實施例中，TACR包含LTβR、HER2、PDL-1、PD-1、EGFR、VEGFR、VEGF、CCR8、OX-40、418B、血管生成素-2、IL-4Ra、BCMA、Blys、BTNO2、C5、CD122、CD13、CD133、CD137、CD138、CD16a、CD19、CD20、CD22、CD27、CD28、CD3、CD30、CD33、CD38、CD40、CD47、CD-8、CEA、CGPR/CGRPR、CSPGs、CTLA4、CTLA-4、DLL-4、EpCAM、因子IXa、因子X、GITR、GP130、Her3、HSG、ICOS、IGFl、IGFl/2、IGF-lR、IGF2、IGFR、IL-1、IL- 12、IL-12p40、IL-13、IL-l 7A、IL-1~、IL-23、IL-5、IL-6、IL-6R、Lag-3、LAG3、MAG、Met、NgR、NogoA、OMGp、OX40、PDGFR、PSMA、RGMA、RGMB、SARS-CoV-2、Te38、TIM-3、TNF、TNFa、TROP-2、TWEAK、或TRAIL。In some embodiments, the second binding portion binds to and/or activates a second target. In some embodiments, the second target is a tumor-associated cell receptor (TACR). TACRs can be expressed on tumor cells or on immune cells associated with tumor cells or the tumor microenvironment. In some embodiments, the second binding portion binds to the TACR. In some embodiments, the second binding portion activates the TACR. In some embodiments, TACR includes LTβR, HER2, PDL-1, PD-1, EGFR, VEGFR, VEGF, CCR8, OX-40, 418B, angiopoietin-2, IL-4Ra, BCMA, Blys, BTNO2, C5, CD122, CD13, CD133, CD137, CD138, CD16a, CD19, CD20, CD22, CD27, CD28, CD3, CD30, CD33, CD38, CD40, CD47, CD-8, CEA, CGPR/CGRPR, CSPGs, CTLA4, CTLA-4, DLL-4, EpCAM, Factor IXa, Factor X, GITR, GP130, Her3, HSG, ICOS, IGF1, IGF1/2, IGF-1R, IGF2, IGFR, IL-1, IL- 12. IL-12p40, IL-13, IL-1 7A, IL-1~, IL-23, IL-5, IL-6, IL-6R, Lag-3, LAG3, MAG, Met, NgR, NogoA, OMGp, OX40, PDGFR, PSMA, RGMA, RGMB, SARS-CoV-2, Te38, TIM-3, TNF, TNFa, TROP-2, TWEAK, or TRAIL.

第二結合部份可係第二抗原結合部份，諸如額外的抗體片段，或可係影響活性的結合多肽或分子，諸如可刺激免疫細胞的細胞介素部份。在一些實施例中，第二結合部份係可結合及/或活化TACR（例如LTβR）之細胞介素部份。The second binding moiety may be a second antigen-binding moiety, such as an additional antibody fragment, or it may be a binding polypeptide or molecule that affects activity, such as a cytokine moiety that can stimulate immune cells. In some embodiments, the second binding moiety is a cytokine moiety that can bind to and/or activate TACR (e.g., LTβR).

在一些實施例中，第二結合部份包含第一部分及第二部分，其中第一部分及第二部分各包含一或多個單元。在一些實施例中，一或多個單元包含第一單元、第二單元、及/或第三單元。在一些實施例中，一或多個單元之各者係相同的。在一些實施例中，一或多個單元係不相同的。在一些實施例中，第二結合部份包含一或多個單元。其中一或多個單元之各者獨立地係腫瘤壞死因子、介白素、淋巴激素、干擾素、群落刺激因子、趨化因子、或生長因子。在一些實施例中，一或多個單元形成作用為第二結合部份的錯合物，替代地，取決於所選擇之第二結合部份，一或多個單元各自個別作用為第二結合部份。第一單元、第二單元、或第三單元之各者可個別包含 表 10所述之蛋白質中之任一者。例如，第一單元、第二單元、或第三單元之各者個別包含LIGHT單元、淋巴毒素-α單元、或淋巴毒素-β單元。在一些實施例中，一或多個單元之各者獨立地包含與 表 10所述之胺基酸序列中之任一者具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%同一性之胺基酸序列。 In some embodiments, the second binding portion comprises a first portion and a second portion, wherein each of the first and second portions comprises one or more units. In some embodiments, the one or more units comprise a first unit, a second unit, and/or a third unit. In some embodiments, each of the one or more units is identical. In some embodiments, the one or more units are different. In some embodiments, the second binding portion comprises one or more units. Each of the one or more units is independently a tumor necrosis factor, interleukin, lymphokine, interferon, community-stimulating factor, chemotherapeutic factor, or growth factor. In some embodiments, the one or more units form a complex that functions as the second binding portion; alternatively, depending on the selected second binding portion, each of the one or more units individually functions as the second binding portion. Each of the first, second, or third units may individually comprise any of the proteins described in Table 10. For example, each of the first, second, or third units may individually comprise a LIGHT unit, a lymphotoxin-α unit, or a lymphotoxin-β unit. In some embodiments, one or more units independently comprise an amino acid sequence having at least 80 %, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any of the amino acid sequences described in Table 10.

在一些實施例中，第二結合部份位於FAP抗原結合部份之C端。在一些實施例中，第二結合部份係可操作地連接至Fc片段之C端。在一些實施例中，第二結合部份藉由連接子連接至Fc片段。在一些實施例中，第二結合部份結合及/或活化第二目標。在一些實施例中，第二結合部份結合及/或活化腫瘤相關細胞受體。在一些實施例中，第二結合部份係腫瘤壞死因子、介白素、淋巴激素、干擾素、群落刺激因子、趨化因子、或生長因子。 例示性抗FAP抗原結合部份 In some embodiments, the second binding portion is located at the C-terminus of the FAP antigen-binding portion. In some embodiments, the second binding portion is operatively linked to the C-terminus of the Fc fragment. In some embodiments, the second binding portion is linked to the Fc fragment via a linker. In some embodiments, the second binding portion binds to and/or activates a second target. In some embodiments, the second binding portion binds to and/or activates tumor-associated cell receptors. In some embodiments, the second binding portion is tumor necrosis factor, interleukin, lymphokine, interferon, community-stimulating factor, chemokine, or growth factor. Example Anti-FAP Antigen Binding Portion

在一些實施例中，FAP抗原結合部份包括上文所描述之抗FAP抗體或其抗原結合片段的部分或全長。在一些實施例中，FAP抗原結合部份包括具有如KTNQNVDYX 1GNTFMH (SEQ ID NO: 23)所示之胺基酸序列的LCDR1（其中X 1係N或S）、具有如LASNLAS (SEQ ID NO: 24)所示之胺基酸序列的LCDR2、具有如QQSRNLPYT (SEQ ID NO: 25)所示之胺基酸序列的LCDR3；具有如IYGVN (SEQ ID NO: 26)所示之胺基酸序列的HCDR1、具有如AIWSGGRKDYX 2LSLKS (SEQ ID NO: 27)所示之胺基酸序列的HCDR2（其中X 2係N或S）、具有如SQDMPGYFDY (SEQ ID NO: 28)所示之胺基酸序列的HCDR3。在一些實施例中，FAP抗原結合部份包括具有如SASSRVGYMH (SEQ ID NO: 29)所示之胺基酸序列的LCDR1、具有如DTSKLAS (SEQ ID NO: 30)所示之胺基酸序列的LCDR2、具有如FQGSGYPFT (SEQ ID NO: 31)所示之胺基酸序列的LCDR3；具有如TAGMSVG (SEQ ID NO: 32)所示之胺基酸序列的HCDR1、具有如DIWWDDKKHYNPSLKD (SEQ ID NO: 33)所示之胺基酸序列的HCDR2、具有如DMIFNFYFDV (SEQ ID NO: 34)所示之胺基酸序列的HCDR3。在一些實施例中，FAP抗原結合部份包括與上文所描述之抗FAP抗體或其抗原結合片段的VH及/或VL相同的VH及/或VL。 In some embodiments, the FAP antigen-binding portion includes part or the full length of the anti-FAP antibody or its antigen-binding fragment as described above. In some embodiments, the FAP antigen binding portion includes LCDR1 (where X1 is N or S) having an amino acid sequence as shown in KTNQNVDYX 1 GNTFMH (SEQ ID NO: 23), LCDR2 having an amino acid sequence as shown in LASSNLAS (SEQ ID NO: 24), LCDR3 having an amino acid sequence as shown in QQSRNLPYT (SEQ ID NO: 25); HCDR1 having an amino acid sequence as shown in IYGVN (SEQ ID NO: 26), HCDR2 (where X2 is N or S) having an amino acid sequence as shown in AIWSGGRKDYX 2 LSLKS (SEQ ID NO: 27), and HCDR3 having an amino acid sequence as shown in SQDMPGYFDY (SEQ ID NO: 28). In some embodiments, the FAP antigen-binding portion includes LCDR1 having an amino acid sequence as shown in SASSRVGYMH (SEQ ID NO: 29), LCDR2 having an amino acid sequence as shown in DTSKLAS (SEQ ID NO: 30), LCDR3 having an amino acid sequence as shown in FQGSGYPFT (SEQ ID NO: 31); HCDR1 having an amino acid sequence as shown in TAGMSVG (SEQ ID NO: 32), HCDR2 having an amino acid sequence as shown in DIWWDDKKHYNPSLKD (SEQ ID NO: 33), and HCDR3 having an amino acid sequence as shown in DMIFNFYFDV (SEQ ID NO: 34). In some embodiments, the FAP antigen-binding portion includes VH and/or VL identical to the VH and/or VL of the anti-FAP antibody or its antigen-binding fragment described above.

在一些實施例中，雙特異性抗體或其抗原結合片段包括第一重鏈、第二重鏈、及分別與第一重鏈及第二重鏈配對的兩條輕鏈，其中(1)第一重鏈包含與SEQ ID NO: 5所述之胺基酸序列具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%同一性之胺基酸序列；該第二重鏈包含與SEQ ID NO: 6所述之胺基酸序列具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%同一性之胺基酸序列；該輕鏈包含與SEQ ID NO: 3所述之胺基酸序列具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%同一性之胺基酸序列；(2)該第一重鏈包含與SEQ ID NO: 8所述之胺基酸序列具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%同一性之胺基酸序列；該第二重鏈包含與SEQ ID NO: 9所述之胺基酸序列具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%同一性之胺基酸序列；該輕鏈包含與SEQ ID NO: 7所述之胺基酸序列具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%同一性之胺基酸序列；(3)該第一重鏈包含與SEQ ID NO: 10所述之胺基酸序列具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%同一性之胺基酸序列；該第二重鏈包含與SEQ ID NO: 11所述之胺基酸序列具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%同一性之胺基酸序列；該輕鏈包含與SEQ ID NO: 3所述之胺基酸序列具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%同一性之胺基酸序列。In some embodiments, the bispecific antibody or its antigen-binding fragment comprises a first heavy chain, a second heavy chain, and two light chains respectively paired with the first heavy chain and the second heavy chain, wherein (1) the first heavy chain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with the amino acid sequence described in SEQ ID NO: 5; the second heavy chain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with the amino acid sequence described in SEQ ID NO: 6; the light chain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with the amino acid sequence described in SEQ ID NO: 6; 3. The amino acid sequence described herein has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity with the amino acid sequence described in SEQ ID NO: 8; (2) The first heavy chain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity with the amino acid sequence described in SEQ ID NO: 9; The second heavy chain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity with the amino acid sequence described in SEQ ID NO: 9; The light chain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity with the amino acid sequence described in SEQ ID NO: 9; 7. The amino acid sequence described herein has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity with the amino acid sequence described in SEQ ID NO: 10; (3) The first heavy chain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity with the amino acid sequence described in SEQ ID NO: 10; the second heavy chain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity with the amino acid sequence described in SEQ ID NO: 11; the light chain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity with the amino acid sequence described in SEQ ID NO: 11; The amino acid sequence described in 3 has an amino acid sequence identity of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%.

在一些實施例中，雙特異性抗體或其抗原結合片段包括具有SEQ ID NO: 5所述之胺基酸序列的第一重鏈、具有SEQ ID NO: 6所述之胺基酸序列的第二重鏈、及具有SEQ ID NO: 3所述之胺基酸序列的輕鏈。在一些實施例中，雙特異性抗體或其抗原結合片段包括具有SEQ ID NO: 8所述之胺基酸序列的第一重鏈、具有SEQ ID NO: 9所述之胺基酸序列的第二重鏈、及具有SEQ ID NO: 7所述之胺基酸序列的輕鏈。在一些實施例中，雙特異性抗體或其抗原結合片段包括具有SEQ ID NO: 10所述之胺基酸序列的第一重鏈、具有SEQ ID NO: 11所述之胺基酸序列的第二重鏈、及具有SEQ ID NO: 3所述之胺基酸序列的輕鏈。In some embodiments, the bispecific antibody or its antigen-binding fragment comprises a first heavy chain having the amino acid sequence of SEQ ID NO: 5, a second heavy chain having the amino acid sequence of SEQ ID NO: 6, and a light chain having the amino acid sequence of SEQ ID NO: 3. In some embodiments, the bispecific antibody or its antigen-binding fragment comprises a first heavy chain having the amino acid sequence of SEQ ID NO: 8, a second heavy chain having the amino acid sequence of SEQ ID NO: 9, and a light chain having the amino acid sequence of SEQ ID NO: 7. In some embodiments, the bispecific antibody or its antigen-binding fragment comprises a first heavy chain having the amino acid sequence of SEQ ID NO: 10, a second heavy chain having the amino acid sequence of SEQ ID NO: 11, and a light chain having the amino acid sequence of SEQ ID NO: 3.

FAP抗原結合部份之另外CDR、VH、VL、HC、及/或LC在本文的FAP結合劑部分中詳細描述。The other CDR, VH, VL, HC, and/or LC of the FAP antigen-binding portion are described in detail in the FAP binder section of this document.

在本揭露中，雙特異性抗體或單株抗體可包括經保守修飾的變體，諸如FR區。經保守修飾的變體包括多肽序列的個別取代、缺失、或添加，這導致胺基酸經化學上相似的胺基酸取代。提供功能相似胺基酸的保守性取代表係所屬技術領域中熟知的且在本文進一步描述。本文進一步描述其他胺基酸修飾。 例示性結構 In this disclosure, bispecific antibodies or monoclonal antibodies may include conserved variants, such as the FR region. Conserved variants include individual substitutions, deletions, or additions of the polypeptide sequence, resulting in the substitution of an amino acid with a chemically similar amino acid. Conserved substitutions of functionally similar amino acids are well known in the art and are further described herein. Other amino acid modifications are further described herein. Illustrative Structures

在一些實施例中，本文所提供之雙特異性抗體包含能夠靶向及結合二或更多個目標的一或多種組分的結構及/或構型。例如，雙特異性抗體可包含如WO2024/193705中所描述的結構A-E中之任一者，該文獻以引用方式全部併入本文中。In some embodiments, the bispecific antibodies provided herein comprise structures and/or configurations of one or more components capable of targeting and binding two or more targets. For example, a bispecific antibody may comprise any of structures A-E as described in WO2024/193705, which is incorporated herein by reference in its entirety.

在一些實施例中，本文所提供之雙特異性抗體包含4鏈抗體單元，該4鏈抗體單元包含兩個H鏈對及兩個L鏈對，且一或多對多肽連接子及第二結合部份附接至各H鏈的Fc區，其中H鏈的胺基酸序列係不相同的且L鏈的胺基酸序列係相同的。在一些實施例中，本文所述之雙特異性抗體包含附接至第一重鏈之FC區的第一多肽連接子及第二結合部份對，且包含附接至第二重鏈之FC區的第二多肽連接子及第二結合部份對、及附接至第二連接子及結合部份對的第三多肽連接子及第二結合部份對。在一些實施例中，本文所述之雙特異性抗體包含附接至第二重鏈之FC區的第一多肽連接子及第二結合部份對，且包含附接至第一重鏈之FC區的第二多肽連接子及第二結合部份對、及附接至第二連接子及結合部份對的第三多肽連接子及第二結合部份對。In some embodiments, the bispecific antibody provided herein comprises a 4-strand antibody unit comprising two H-strand pairs and two L-strand pairs, and one or more pairs of polypeptide linkers and second binding moieties attached to the Fc region of each H-strand, wherein the amino acid sequences of the H-strands are different and the amino acid sequences of the L-strands are identical. In some embodiments, the bispecific antibody described herein comprises a first polypeptide linker and a second binding moieties attached to the FC region of a first heavy chain, and comprises a second polypeptide linker and a second binding moieties attached to the FC region of a second heavy chain, and a third polypeptide linker and a second binding moieties attached to the second linker and binding moieties. In some embodiments, the bispecific antibody described herein includes a first polypeptide linker and a second binding moiety pair attached to the FC region of the second heavy chain, and includes a second polypeptide linker and a second binding moiety pair attached to the FC region of the first heavy chain, and a third polypeptide linker and a second binding moiety pair attached to the second linker and the binding moiety pair.

本揭露的雙特異性抗體可係雙可變域免疫球蛋白(DVD-Ig ™)，如Jakob, C. G., Edalji, R., Judge, R. A., DiGiammarino, E., Li, Y., Gu, J., & Ghayur, T. (2013).Structure reveals function of the dual variable domain immunoglobulin (DVD-IgTM) molecule. mAbs, 5(3), 358–363中所述，其經由可撓性的天然存在的連接子組合兩個單株抗體的目標結合域，此產出四價IgG樣分子。 The bispecific antibody disclosed herein may be a dual variable domain immunoglobulin (DVD-Ig ™ ), as described in Jakob, CG, Edalji, R., Judge, RA, DiGiammarino, E., Li, Y., Gu, J., & Ghayur, T. (2013). Structure reveals function of the dual variable domain immunoglobulin (DVD-Ig™) molecule. mAbs, 5(3), 358–363, which combines the target binding domains of two monoclonal antibodies via a flexible, naturally occurring linker, thus producing a tetravalent IgG-like molecule.

在一些實施例中，雙特異性抗體或其抗原結合片段具有如圖2或圖6所示的形式D。雙特異性抗體或其抗原結合片段具有如圖2或圖6所示的形式D。 細胞介素部份 In some embodiments, the bispecific antibody or its antigen-binding fragment has form D as shown in Figure 2 or Figure 6. The bispecific antibody or its antigen-binding fragment has form D as shown in Figure 2 or Figure 6. Intercytokine portion

在一些實施例中，本揭露係關於一種雙特異性抗體或其抗原結合片段，其包括FAP抗原結合部份及能夠刺激免疫細胞的細胞介素部份。細胞介素部份可係任何能夠刺激免疫細胞的分子。細胞介素部份可在雙特異性抗體或其抗原結合片段之C端。在一些實施例中，細胞介素部份係可操作地連接至雙特異性抗體或其抗原結合片段之Fc片段之C端。在一些實施例中，細胞介素部份直接連接至Fc片段。在一些實施例中，細胞介素部份經由連接子連接至Fc片段。本文進一步描述例示性連接子。In some embodiments, this disclosure relates to a bispecific antibody or an antigen-binding fragment thereof, comprising an FAP antigen-binding portion and a cytokine portion capable of stimulating immune cells. The cytokine portion can be any molecule capable of stimulating immune cells. The cytokine portion may be located at the C-terminus of the bispecific antibody or its antigen-binding fragment. In some embodiments, the cytokine portion is operatively linked to the C-terminus of the Fc fragment of the bispecific antibody or its antigen-binding fragment. In some embodiments, the cytokine portion is directly linked to the Fc fragment. In some embodiments, the cytokine portion is linked to the Fc fragment via a linker. Exemplary linkers are further described herein.

在一些實施例中，細胞介素部份包括第一細胞介素部份及第二細胞介素部份。第一細胞介素部份及第二細胞介素部份之各者可包含第一細胞介素單元、第二細胞介素單元、及/或第三細胞介素單元。在一些實施例中，第一細胞介素部份包含第一細胞介素單元。在一些實施例中，第一細胞介素單元包含LIGHT單元或淋巴毒素-β單元。在一些實施例中，第二細胞介素部份包含第二細胞介素單元及第三細胞介素單元。在一些實施例中，第二細胞介素單元包含LIGHT單元、淋巴毒素-α單元、或淋巴毒素-β單元。在一些實施例中，第三細胞介素單元包含LIGHT單元、淋巴毒素-α單元、或淋巴毒素-β單元。在一些實施例中，第一細胞介素部份包含第一LIGHT單元，第二細胞介素單元包含第二LIGHT單元，且第三細胞介素單元包含第三LIGHT單元。在一些實施例中，第一LIGHT單元、第二LIGHT單元、及/或第三LIGHT單元各自獨立地包含SEQ ID NO: 17或18所述之胺基酸序列。在一些實施例中，第一細胞介素部份包含淋巴毒素-β單元，第二細胞介素單元包含淋巴毒素-α單元，且第三細胞介素單元包含淋巴毒素-β單元。在一些實施例中，淋巴毒素-β單元包含SEQ ID NO: 39所述之胺基酸序列，淋巴毒素-α單元包含SEQ ID NO: 40所述之胺基酸序列。In some embodiments, the intercytokine portion includes a first intercytokine portion and a second intercytokine portion. Each of the first and second intercytokine portions may contain a first intercytokine unit, a second intercytokine unit, and/or a third intercytokine unit. In some embodiments, the first intercytokine portion contains a first intercytokine unit. In some embodiments, the first intercytokine unit contains a LIGHT unit or a lymphotoxin-β unit. In some embodiments, the second intercytokine portion includes a second intercytokine unit and a third intercytokine unit. In some embodiments, the second intercytokine unit contains a LIGHT unit, a lymphotoxin-α unit, or a lymphotoxin-β unit. In some embodiments, the third intercellular unit comprises a LIGHT unit, a lymphotoxin-α unit, or a lymphotoxin-β unit. In some embodiments, the first intercellular portion comprises a first LIGHT unit, the second intercellular unit comprises a second LIGHT unit, and the third intercellular unit comprises a third LIGHT unit. In some embodiments, the first LIGHT unit, the second LIGHT unit, and/or the third LIGHT unit each independently comprises the amino acid sequence described in SEQ ID NO: 17 or 18. In some embodiments, the first intercellular portion comprises a lymphotoxin-β unit, the second intercellular unit comprises a lymphotoxin-α unit, and the third intercellular unit comprises a lymphotoxin-β unit. In some embodiments, the lymphotoxin-β unit comprises the amino acid sequence described in SEQ ID NO: 39, and the lymphotoxin-α unit comprises the amino acid sequence described in SEQ ID NO: 40.

在一些實施例中，細胞介素部份包括第一細胞介素部份及第二細胞介素部份，第一細胞介素部份含有一個LIGHT突變體，且第二細胞介素部份含有串聯連接的兩個LIGHT突變體。In some embodiments, the intercytokine portion includes a first intercytokine portion and a second intercytokine portion, the first intercytokine portion containing a LIGHT mutant and the second intercytokine portion containing two LIGHT mutants connected in series.

在一些實施例中，細胞介素部份由一或多個連接子連接至Fc片段。在一些實施例中，第一細胞介素部份係藉由連接子連接至Fc片段。在一些實施例中，第二細胞介素部份係藉由連接子連接至Fc片段。在一些實施例中，第一細胞介素部份及/或第二細胞介素部份之細胞介素單元之各者由一或多個連接子連接。在一些實施例中，第一細胞介素部份經由連接子A連接至Fc片段，且第二細胞介素部份經由連接子B連接至Fc片段。在一些實施例中，串聯連接的兩個LIGHT突變體直接或經由連接子C彼此連接。在一些實施例中，連接子A、連接子B、及連接子C獨立地係具有式(Gly4Ser) n的肽連接子，其中n係1、2、3、4、或5。在一些實施例中，n係2或3，亦即，連接子係(Gly4Ser) 2或(Gly4Ser) 3。在一些實施例中，連接子A、連接子B、及連接子C獨立地係胺基酸G。本文進一步描述例示性連接子。 In some embodiments, the intercytokine portion is linked to the Fc fragment by one or more linkers. In some embodiments, the first intercytokine portion is linked to the Fc fragment by a linker. In some embodiments, the second intercytokine portion is linked to the Fc fragment by a linker. In some embodiments, each of the intercytokine units of the first and/or second intercytokine portions is linked by one or more linkers. In some embodiments, the first intercytokine portion is linked to the Fc fragment via linker A, and the second intercytokine portion is linked to the Fc fragment via linker B. In some embodiments, two LIGHT mutants connected in series are linked to each other directly or via linker C. In some embodiments, linkers A, B, and C are independently peptide linkers having the formula (Gly4Ser) n , where n is 1, 2, 3, 4, or 5. In some embodiments, n is 2 or 3, that is, the linker is (Gly4Ser) 2 or (Gly4Ser) 3 . In some embodiments, linkers A, B, and C are independently amino acid G. Exemplary linkers are further described herein.

在一些實施例中，細胞介素部份結合及/或活化TACR。在一些實施例中，細胞介素部份結合及/或活化LTβR。在一些實施例中，細胞介素部份結合及/或活化TACR，該TACR包含與 表 5.1所述之胺基酸序列中之任一者具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%同一性之胺基酸序列。在一些實施例中，第一細胞介素部份結合及/或活化TACR，該TACR包含與 表 5.1所述之胺基酸序列中之任一者具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%同一性之胺基酸序列。在一些實施例中，第二細胞介素部份結合及/或活化TACR，該TACR包含與 表 5.1所述之胺基酸序列中之任一者具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%同一性之胺基酸序列。在一些實施例中，第一細胞介素部份及第二細胞介素部份包含各自個別或共同結合及/或活化TACR之一或多個單元，該TACR包含與 表 5.1所述之胺基酸序列中之任一者具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%同一性之胺基酸序列。 In some embodiments, the intercytokine partially binds to and/or activates the TACR. In some embodiments, the intercytokine partially binds to and/or activates the LTβR. In some embodiments, the intercytokine partially binds to and/or activates the TACR, which comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any of the amino acid sequences described in Table 5.1 . In some embodiments, a first intercytokine partially binds to and/or activates the TACR, which comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any of the amino acid sequences described in Table 5.1. In some embodiments, the second interleukin moiety binds to and/or activates a TACR containing an amino acid sequence having at least 80%, 85 % , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any of the amino acid sequences described in Table 5.1 . In some embodiments, the first and second interleukin moieties each individually or jointly bind to and/or activate one or more units of a TACR containing an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any of the amino acid sequences described in Table 5.1.

在一些實施例中，細胞介素部份包含腫瘤壞死因子、介白素、淋巴激素、干擾素、群落刺激因子、趨化因子、或生長因子。在一些實施例中，腫瘤壞死因子包含LIGHT、淋巴毒素α、淋巴毒素β、或4-1 BBL、或其組合。在一些實施例中，細胞介素部份包含一或多個LIGHT、淋巴毒素α、淋巴毒素β、或4-1 BBL、或其組合。In some embodiments, the intercytokine portion comprises tumor necrosis factor, interleukin, lymphokine, interferon, community-stimulating factor, chemokine, or growth factor. In some embodiments, the tumor necrosis factor comprises LIGHT, lymphotoxin α, lymphotoxin β, or 4-1 BBL, or combinations thereof. In some embodiments, the intercytokine portion comprises one or more LIGHT, lymphotoxin α, lymphotoxin β, or 4-1 BBL, or combinations thereof.

在一些實施例中，細胞介素部份包含與 表 10所述之胺基酸序列中之任一者具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%同一性或與其同一之胺基酸序列。在一些實施例中，第一細胞介素部份包含與 表 10所述之胺基酸序列中之任一者具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%同一性或與其同一之胺基酸序列。在一些實施例中，第二細胞介素部份包含與 表 10所述之胺基酸序列中之任一者具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%同一性或與其同一之胺基酸序列。在一些實施例中，第一細胞介素部份及第二細胞介素部份包含一或多個單元，其中各單元包含與 表 10所述之胺基酸序列中之任一者具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%同一性或與其同一之胺基酸序列。 In some embodiments , the intercellular element portion comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 % , 98%, or 99% identity with or identical to any of the amino acid sequences described in Table 10. In some embodiments, the first intercellular element portion comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with or identical to any of the amino acid sequences described in Table 10. In some embodiments, the second intercellular element portion comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with or identical to any of the amino acid sequences described in Table 10. In some embodiments, the first intercytokine portion and the second intercytokine portion comprise one or more units, wherein each unit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with or the same as any of the amino acid sequences described in Table 10.

在一些實施例中，細胞介素部份係腫瘤相關細胞受體配體。在一些實施例中，細胞介素部份係LIGHT單元。在一些實施例中，細胞介素部份係LIGHT突變體。在一些實施例中，第一細胞介素部份係LIGHT突變體。在一些實施例中，第二細胞介素部份係LIGHT突變體。在一些實施例中，第一細胞介素部份及第二細胞介素部份包含一或多個單元，其中各單元係LIGHT突變體或LIGHT突變體之一部分。在一些實施例中，LIGHT突變體包括SEQ ID NO: 17或18所述之胺基酸序列。在一些實施例中，第一細胞介素部份包含一個淋巴毒素-β突變體，且第二細胞介素部份包含串聯連接的淋巴毒素-αβ突變體，淋巴毒素-β突變體包括如SEQ ID NO: 39所示之胺基酸序列，淋巴毒素-α突變體包含如SEQ ID NO: 40所示之胺基酸序列。In some embodiments, the interleukin portion is a tumor-associated cell receptor ligand. In some embodiments, the interleukin portion is a LIGHT unit. In some embodiments, the interleukin portion is a LIGHT mutant. In some embodiments, the first interleukin portion is a LIGHT mutant. In some embodiments, the second interleukin portion is a LIGHT mutant. In some embodiments, the first and second interleukin portions comprise one or more units, wherein each unit is a LIGHT mutant or a portion thereof. In some embodiments, the LIGHT mutant comprises the amino acid sequence described in SEQ ID NO: 17 or 18. In some embodiments, the first intercellular portion includes a lymphotoxin-β mutant, and the second intercellular portion includes tandemly linked lymphotoxin-αβ mutants, the lymphotoxin-β mutant including the amino acid sequence shown in SEQ ID NO: 39, and the lymphotoxin-α mutant including the amino acid sequence shown in SEQ ID NO: 40.

在一些實施例中，細胞介素部份包含與WO2024/193705（其以引用方式全部併入本文中）中描述之細胞介素部份胺基酸序列中之任一者具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%同一性或與其同一之至少一個胺基酸序列。在一些實施例中，細胞介素部份之各單元包含與WO2024/193705（其以引用方式全部併入本文中）中描述之細胞介素部份胺基酸序列中之任一者具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%同一性或與其同一之胺基酸序列。In some embodiments, the interleukin portion includes at least one amino acid sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with or is identical to any of the amino acid sequences of the interleukin portion described in WO2024/193705 (which is incorporated herein by reference in its entirety). In some embodiments, each unit of the interleukin portion includes an amino acid sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with or is identical to any of the amino acid sequences of the interleukin portion described in WO2024/193705 (which is incorporated herein by reference in its entirety).

如所描述的，雙特異性抗體或其抗原結合片段可觸發用於癌相關纖維母細胞(cancer-associated fibroblast, CAF)重編程的LTβR信號傳導。不受理論的束縛，據信FAP以順式或反式方式接合雙特異性抗體或其抗原結合片段將增強LTβR路徑活化的效率。As described, bispecific antibodies or their antigen-binding fragments can trigger LTβR signaling for cancer-associated fibroblast (CAF) reprogramming. Regardless of theoretical constraints, it is believed that FAP binding to bispecific antibodies or their antigen-binding fragments in either a cis or trans manner will enhance the efficiency of LTβR pathway activation.

LTβR信號傳導對於驅動第二淋巴器官(2nd lymphoid organ, SLO)的形成至關重要，且在一些情況下，對於驅動三級淋巴結構(TLS)的形成亦至關重要。本揭露旨在觸發FAP+CAF（癌相關纖維母細胞）中的LTβR信號傳導，且將其重新程式化為TLS促進表型，因此增強抗腫瘤免疫性。亦據信，在具有高度特異性LTβR靶向的腫瘤基質細胞上表現的交聯FAP選擇性地靶向LTβR且將LTβR促效作用專門限制在腫瘤微環境（腫瘤內皮及癌相關纖維母細胞）中，藉以減少潛在的副作用。LTβR signaling is crucial for driving the formation of the second lymphoid organ (SLO) and, in some cases, for driving the formation of tertiary lymphoid structures (TLS). This disclosure aims to trigger LTβR signaling in FAP+CAF (cancer-associated fibroblasts) and reprogram it into a TLS-promoting phenotype, thereby enhancing antitumor immunity. It is also believed that cross-linked FAPs expressed on tumor stromal cells with high LTβR targeting selectively target LTβR and specifically restrict the LTβR-promoting effect to the tumor microenvironment (tumor endothelial cells and cancer-associated fibroblasts), thereby reducing potential side effects.

在一些實施例中，例如使用平台D產生的雙特異性抗體或其抗原結合片段能夠穩健地產生免疫細胞介素，表現出有前景的抗腫瘤功效。In some embodiments, for example, bispecific antibodies or antigen-binding fragments of antibodies produced by Platform D can robustly generate immunocytokines, demonstrating promising antitumor efficacy.

在一些實施例中，雙特異性抗體或其抗原結合片段以不大於20 nM、15 nM、10 nM、或5 nM之解離常數（KD，KD=koff/kon，或KD= Kd/Ka）結合至FAP。在一些實施例中，雙特異性抗體或其抗原結合片段以不大於20 nM、15 nM、10 nM、或5 nM之解離常數(KD)結合至LTßR。在一些實施例中，雙特異性抗體或其抗原結合片段幾乎不結合至人類或食蟹獼猴HVEM。在一些實施例中，LIGHT突變體能夠降低與DcR3之結合親和力。在一些實施例中，雙特異性抗體或其抗原結合片段特異性結合至人類FAP，且/或不結合至DPPIV。本文進一步描述雙特異性抗體之活性。In some embodiments, the bispecific antibody or its antigen-binding fragment binds to FAP with a dissociation constant (KD, KD = koff/kon, or KD = Kd/Ka) not greater than 20 nM, 15 nM, 10 nM, or 5 nM. In some embodiments, the bispecific antibody or its antigen-binding fragment binds to LTßR with a dissociation constant (KD) not greater than 20 nM, 15 nM, 10 nM, or 5 nM. In some embodiments, the bispecific antibody or its antigen-binding fragment hardly binds to human or cynomolgus monkey HVEM. In some embodiments, the LIGHT mutant can reduce the binding affinity to DcR3. In some embodiments, the bispecific antibody or its antigen-binding fragment specifically binds to human FAP and/or does not bind to DPPIV. The activity of the bispecific antibody is further described herein.

在一些實施例中，雙特異性抗體或其抗原結合片段包含融合蛋白。在一些實施例中，雙特異性抗體或其抗原結合片段包含抗FAP融合蛋白。在一些實施例中，雙特異性抗體或其抗原結合片段包含抗FAP細胞介素融合蛋白。在一些實施例中，雙特異性抗體或其抗原結合片段包含抗FAP-LIGHT融合蛋白。在一些實施例中，雙特異性抗體或抗原結合片段包含抗FAP-淋巴毒素-αββ融合蛋白。在一些實施例中，雙特異性抗體或其抗原結合片段係抗FAP-LIGHT融合蛋白。在一些實施例中，雙特異性抗體或其抗原結合片段係抗FAP-淋巴毒素-αββ融合蛋白。在一些實施例中，融合蛋白包括雙特異性抗體。本文進一步描述融合蛋白。 第二抗原結合部份 In some embodiments, the bispecific antibody or its antigen-binding fragment comprises a fusion protein. In some embodiments, the bispecific antibody or its antigen-binding fragment comprises an anti-FAP fusion protein. In some embodiments, the bispecific antibody or its antigen-binding fragment comprises an anti-FAP interleukin fusion protein. In some embodiments, the bispecific antibody or its antigen-binding fragment comprises an anti-FAP-LIGHT fusion protein. In some embodiments, the bispecific antibody or its antigen-binding fragment comprises an anti-FAP-lymphotoxin-αββ fusion protein. In some embodiments, the bispecific antibody or its antigen-binding fragment is an anti-FAP-LIGHT fusion protein. In some embodiments, the bispecific antibody or its antigen-binding fragment is an anti-FAP-lymphotoxin-αββ fusion protein. In some embodiments, the fusion protein comprises a bispecific antibody. This article further describes the fusion protein. Second antigen-binding portion

在一些實施例中，本文所提供之FAP結合劑之FAP結合部分係與第二抗體及/或其抗原結合片段、第二抗原結合部份、或其構築體連接、接合、或融合。In some embodiments, the FAP-binding portion of the FAP binder provided herein is linked, conjugated, or fused with a second antibody and/or its antigen-binding fragment, second antigen-binding portion, or its building block.

在一些實施例中，本文所提供之FAP結合劑之FAP結合部分係與第二抗體連接、接合、或融合以形成抗體異源接合物。在一些實施例中，本文所提供之FAP結合劑之FAP結合部分係與第二結合部份連接、接合、或融合，產生多特異性抗體。多特異性抗體（諸如雙特異性抗體）係對至少兩個不同目標（例如抗原）或同一目標上的兩個不同表位（例如針對FAP的雙特異性抗體，其具有針對FAP的第一表位的第一結合部份及針對FAP的第二表位的第二結合部份）具有結合特異性的單株抗體。在一些實施例中，多特異性（例如雙特異性）抗體可基於本文所述之抗體的序列（例如 表 6及 表 7中的CDR序列）建構。在一些實施例中，本文所述之多特異性抗體係雙特異性抗體。在一些實施例中，雙特異性抗體係小鼠抗體、嵌合抗體、人類抗體、或人源化抗體。在一些實施例中，多特異性抗體的結合特異性中的一者係針對FAP，且另一者係針對任何其他目標（例如抗原）。在一些實施例中，多特異性（例如雙特異性）抗體可包含多於一個的目標（例如抗原）結合部份，其中不同的結合部份對不同的目標具有特異性（例如結合至FAP的第一結合部份及結合另一目標（例如抗原）的第二結合部份，諸如免疫檢查點調節劑（例如負檢查點調節劑）。在一些實施例中，多特異性（例如雙特異性）抗體分子可結合相同目標（例如抗原）上的多於一個（例如二或更多個）表位。例如，第二結合部份可係衍生自本文進一步描述之額外治療性抗體之任一者的部份。 In some embodiments, the FAP-binding portion of the FAP conjugate provided herein is linked, conjugated, or fused with a second antibody to form an antibody heteroconjugate. In some embodiments, the FAP-binding portion of the FAP conjugate provided herein is linked, conjugated, or fused with a second binding portion to produce a multispecific antibody. Multispecific antibodies (such as bispecific antibodies) are monoclonal antibodies that have binding specificity to at least two different targets (e.g., antigens) or two different epitopes on the same target (e.g., a bispecific antibody against FAP having a first binding portion against a first epitope of FAP and a second binding portion against a second epitope of FAP). In some embodiments, multispecific (e.g., bispecific) antibodies may be constructed based on the sequences of the antibodies described herein (e.g., the CDR sequences in Tables 6 and 7 ). In some embodiments, the multispecific antibodies described herein are bispecific antibodies. In some embodiments, the bispecific antibodies are mouse antibodies, chimeric antibodies, human antibodies, or humanized antibodies. In some embodiments, the binding specificity of the multispecific antibody is one against FAP and the other against any other target (e.g., antigen). In some embodiments, a multispecific (e.g., bispecific) antibody may include more than one target (e.g., antigen) binding moiety, wherein different binding moieties are specific for different targets (e.g., a first binding moiety binding to an FAP and a second binding moiety binding to another target (e.g., an antigen), such as immune checkpoint regulators (e.g., negative checkpoint regulators). In some embodiments, a multispecific (e.g., bispecific) antibody molecule may bind to more than one (e.g., two or more) epitopes on the same target (e.g., antigen). For example, the second binding moiety may be a portion derived from any of the additional therapeutic antibodies described further herein.

在一些實施例中，結合特異性中的一者係針對FAP，且另一者係針對一或多種TACR。TACR可係趨化因子受體、細胞表面蛋白（諸如在免疫細胞上表現的細胞表面蛋白、或組織或細胞類型特異性抗原）、或與T細胞活化相關的細胞表面分子。In some embodiments, one of the binding specificities is for FAP, and the other is for one or more TACRs. TACRs may be chemokine receptors, cell surface proteins (such as cell surface proteins expressed on immune cells, or tissue or cell type-specific antigens), or cell surface molecules associated with T cell activation.

例如，TACR可係腫瘤壞死因子受體，諸如LTβR。在一些實施例中，第二抗體片段係描述於U.S. 6,312,691、WO 96/22788、WO2018119118、WO9622788、WO2006/114284、WO2004/058191、WO02/30986、WO2022117572、及WO2007146414中的抗LTβR抗體，其中之各者的內容特此以引用的方式全部併入本文中。在一些實施例中，第二抗體片段係BKA11、CDH10、BCG6、AGH1、BDA8、CBE11、或BHA10。For example, TACR can be a tumor necrosis factor receptor, such as LTβR. In some embodiments, the second antibody fragment is an anti-LTβR antibody described in U.S. 6,312,691, WO 96/22788, WO2018119118, WO9622788, WO2006/114284, WO2004/058191, WO02/30986, WO2022117572, and WO2007146414, the contents of which are hereby incorporated herein by reference in their entirety. In some embodiments, the second antibody fragment is BKA11, CDH10, BCG6, AGH1, BDA8, CBE11, or BHA10.

在另一實例中，TACR可係趨化因子受體。例示性的不同趨化因子受體包括CCR1、CCR2、CCR3、CCR4、CCR5、CCR6、CCR7、CCR8、CCR9、CCR10、CXCR1、CXCR2、CXCR3、CXCR4、CXCR5、CXCR6、CX3CR1、或CXCR1。例如，雙特異性抗體之第二結合部份係莫格利珠單抗(Mogamulizumab)或其抗原結合片段。In another example, a TACR can be a chemokine receptor. Illustrative chemokine receptors include CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CX3CR1, or CXCR1. For example, the second binding moiety of a bispecific antibody is mogamulizumab or its antigen-binding fragment.

靶向TACR的例示性結合部份包括細胞表面蛋白，諸如在免疫細胞上表現的細胞表面蛋白、或組織或細胞類型特異性抗原。此類結合部份包括靶向檢查點蛋白的抗體或抗原結合片段，諸如抗PD1抗體、抗PD-L1抗體、或CTLA-4抗體。適合的檢查點蛋白靶向抗體包括納武單抗、派姆單抗、阿特珠單抗、阿維魯單抗、德瓦魯單抗、西米普利單抗、多斯利單抗、或伊匹單抗。在一些實施例中，雙特異性抗體之第二結合部份係HER2靶向抗體，諸如曲妥珠單抗、帕妥珠單抗、及/或馬吉妥昔單抗(Margetuximab)。與T細胞活化相關的例示性細胞表面分子包括CD25、CTLA-4、PD-1、LAG3、TIGIT、ICOS、及TNF受體超家族成員4-1BB、OX-40、及GITR。Exemplary binding moieties targeting TACRs include cell surface proteins, such as those expressed on immune cells, or tissue or cell type-specific antigens. These binding moieties include antibody or antigen-binding fragments targeting checkpoint proteins, such as anti-PD1 antibodies, anti-PD-L1 antibodies, or CTLA-4 antibodies. Suitable checkpoint protein-targeting antibodies include nivolumab, pembrolizumab, atezolizumab, averrucumab, durvalumab, cimiprimab, doslizumab, or ipilimumab. In some embodiments, the second binding moieties of bispecific antibodies are HER2-targeting antibodies, such as trastuzumab, pertuzumab, and/or margetuximab. Exemplary cell surface molecules associated with T cell activation include CD25, CTLA-4, PD-1, LAG3, TIGIT, ICOS, and TNF receptor superfamily members 4-1BB, OX-40, and GITR.

適合與本文所提供之FAP抗原結合部份一起使用的其他第二抗原結合片段描述於WO 2020208049、WO 2018178074、WO 2017060144、WO 2018127473、WO 2020007817、WO 2017055398、WO 2023073225、WO 2023025194、WO 2024175105、WO 2023025194、WO 2024175105、WO 2024179567、WO 2019222449、WO 2019086500、WO 2021236658、CN 113307879、WO 2020230899、WO 2020230899、WO 2020230899、WO 2019086497、WO 2022262496、WO 2022262496、WO 2018178074、WO 2017055398、WO 2018185045、WO 2020127628、WO 2020070041、WO 2020070035、WO 2023117834、WO 2021198335、WO 2021198333、WO 2021198335、WO 2023117834、WO 2021257808、WO 2024133330、WO 2023050826、WO 2024199269、WO 2023050826、WO 2024199269、WO 2019195623、N/A、WO-2018178074、WO-2024179567、N/A、N/A、WO-2020245173、WO 2016075278、WO 2023110788、WO 2024184287、WO 2024188966、WO-2014161845、WO 2016055432、WO-2022101458、及WO202413330，其中之各者的內容以引用方式全部併入本文中。此類第二抗原結合片段靶向CD40分子(CD40)、Fc γ受體IIIa (FCGR3A)、細胞毒性T淋巴球相關蛋白4 (CTLA4)、CD28分子(CD28)、CD276分子(CD276)、CD3錯合物（T細胞受體錯合物）、轉化生長因子β受體2 (TGFbR2)，且因此其中之各者均係本文所述之適合的次要目標。Other second antigen-binding fragments suitable for use with the FAP antigen-binding portion provided herein are described in WO 2020208049, WO 2018178074, WO 2017060144, WO 2018127473, WO 2020007817, WO 2017055398, WO 2023073225, WO 2023025194, WO 2024175105, WO 2023025194, WO 2024175105, WO 2024179567, WO 2019222449, WO 2019086500, WO 2021236658, CN 113307879, WO 2020230899, WO 2020230899、WO 2020230899、WO 2019086497、WO 2022262496、WO 2022262496、WO 2018178074、WO 2017055398、WO 2018185045、WO 2020127628、WO 2020070041, WO 2020070035, WO 2023117834, WO 2021198335, WO 2021198333, WO 2021198335, WO 2023117834, WO 2021257808, WO 2024133330, WO The contents of WO 2023050826, WO 2024199269, WO 2023050826, WO 2024199269, WO 2019195623, N/A, WO-2018178074, WO-2024179567, N/A, N/A, WO-2020245173, WO 2016075278, WO 2023110788, WO 2024184287, WO 2024188966, WO-2014161845, WO 2016055432, WO-2022101458, and WO202413330 are all incorporated herein by reference in their entirety. These second antigen-binding fragments target CD40 molecules (CD40), Fc γ receptor IIIa (FCGR3A), cytotoxic T lymphocyte-associated protein 4 (CTLA4), CD28 molecules (CD28), CD276 molecules (CD276), CD3 complexes (T cell receptor complexes), and transforming growth factor β receptor 2 (TGFbR2), and therefore each of these are suitable secondary targets as described herein.

在一些實施例中，第二抗原結合部份係抗LTβR結合部份、抗HER2結合部份、抗PDL-1結合部份、抗PD-1結合部份、抗EGFR結合部份、抗VEGFR結合部份、抗VEGF結合部份、抗CCR8結合部份、抗OX-40結合部份、抗418B結合部份、抗血管生成素-2結合部份、抗IL-4Ra結合部份、抗BCMA結合部份、抗Blys結合部份、抗BTNO2結合部份、抗C5結合部份、抗CD122結合部份、抗CD13結合部份、抗CD133結合部份、抗CD137結合部份、抗CD138結合部份、抗CD16a結合部份、抗CD19結合部份、抗CD20結合部份、抗CD22結合部份、抗CD27結合部份、抗CD28結合部份、抗CD3結合部份、抗CD30結合部份、抗CD33結合部份、抗CD38結合部份、抗CD40結合部份、抗CD47結合部份、抗CD-8結合部份、抗CEA結合部份、抗CGPR/CGRPR結合部份、抗CSPGs結合部份、抗CTLA4結合部份、抗CTLA-4結合部份、抗DLL-4結合部份、抗EpCAM結合部份、抗因子IXa結合部份、抗因子X結合部份、抗GITR結合部份、抗GP130結合部份、抗Her3結合部份、抗HSG結合部份、抗ICOS結合部份、抗IGFl結合部份、抗IGFl/2結合部份、抗IGF-lR結合部份、抗IGF2結合部份、抗IGFR結合部份、抗IL-1結合部份、抗IL-12結合部份、抗IL-12p40結合部份、抗IL-13結合部份、抗IL-l 7A結合部份、抗IL-1~結合部份、抗IL-23結合部份、抗IL-5結合部份、抗IL-6結合部份、抗IL-6R結合部份、抗Lag-3結合部份、抗LAG3結合部份、抗MAG結合部份、抗Met結合部份、抗NgR結合部份、抗NogoA結合部份、抗OMGp結合部份、抗OX40結合部份、抗PDGFR結合部份、抗PSMA結合部份、抗RGMA結合部份、抗RGMB結合部份、抗SARS-CoV-2結合部份、抗Te38結合部份、抗TIM-3結合部份、抗TNF結合部份、抗TNFa結合部份、抗TROP-2結合部份、抗TWEAK結合部份、或抗TRAIL結合部份。In some embodiments, the second antigen-binding moiety is an anti-LTβR binding moiety, an anti-HER2 binding moiety, an anti-PDL-1 binding moiety, an anti-PD-1 binding moiety, an anti-EGFR binding moiety, an anti-VEGFR binding moiety, an anti-VEGF binding moiety, an anti-CCR8 binding moiety, an anti-OX-40 binding moiety, an anti-418B binding moiety, an anti-angiogenic-2 binding moiety, an anti-IL-4Ra binding moiety, an anti-BCMA binding moiety, or an anti- Blys binding site, anti-BTNO2 binding site, anti-C5 binding site, anti-CD122 binding site, anti-CD13 binding site, anti-CD133 binding site, anti-CD137 binding site, anti-CD138 binding site, anti-CD16a binding site, anti-CD19 binding site, anti-CD20 binding site, anti-CD22 binding site, anti-CD27 binding site, anti-CD28 binding site, anti-CD3 binding site, anti-CD3 0-binding region, anti-CD33-binding region, anti-CD38-binding region, anti-CD40-binding region, anti-CD47-binding region, anti-CD-8-binding region, anti-CEA-binding region, anti-CGPR/CGRPR-binding region, anti-CSPGs-binding region, anti-CTLA4-binding region, anti-CTLA-4-binding region, anti-DLL-4-binding region, anti-EpCAM-binding region, anti-factor IXa-binding region, anti-factor X-binding region Anti-GITR binding site, anti-GP130 binding site, anti-Her3 binding site, anti-HSG binding site, anti-ICOS binding site, anti-IGF1 binding site, anti-IGF1/2 binding site, anti-IGF-1R binding site, anti-IGF2 binding site, anti-IGFR binding site, anti-IL-1 binding site, anti-IL-12 binding site, anti-IL-12p40 binding site, anti-IL-13 binding site, anti-IL-1 7A binding region, anti-IL-1 binding region, anti-IL-23 binding region, anti-IL-5 binding region, anti-IL-6 binding region, anti-IL-6R binding region, anti-Lag-3 binding region, anti-LAG3 binding region, anti-MAG binding region, anti-Met binding region, anti-NgR binding region, anti-NogoA binding region, anti-OMGp binding region, anti-OX40 binding region, anti-PDGFR binding region, anti-PSMA binding region, anti-RGMA binding region, anti-RGMB binding region, anti-SARS-CoV-2 binding region, anti-Te38 binding region, anti-TIM-3 binding region, anti-TNF binding region, anti-TNFα binding region, anti-TROP-2 binding region, anti-TWEAK binding region, or anti-TRAIL binding region.

在一些實施例中，本文所提供之雙特異性抗體包含FAP抗原結合部份及靶向第二目標且附接至藥物部份之第二抗原結合部份。在一些實施例中，第二抗原結合部份係薩西土珠單抗(sacituzumab)，藥物部份係戈維特坎(govitecan)，且第二目標係TROP2。在一些實施例中，第二抗原結合部份係泰舒圖單抗(tisotumab)，藥物部份係維多汀(vedotin)，且第二目標係組織因子。在一些實施例中，第二抗原結合部份係因福土單抗(enfortumab)，藥物部份係維多汀(vedotin)，且第二目標係Nectin4。在一些實施例中，第二抗原結合部份係本妥昔單抗(brentuximab)，藥物部份係維多汀(vedotin)，且第二目標係CD30。在一些實施例中，第二抗原結合部份係曲妥珠單抗(trastuzumab)，藥物部份係德魯替康(deruxtecan)，且第二目標係HER2。在一些實施例中，第二抗原結合部份係曲妥珠單抗(trastuzumab)，藥物部份係恩他新(emtansine)，且第二目標係HER2。在一些實施例中，第二抗原結合部份係保納珠單抗(polatuzumab)，藥物部份係維多汀(vedotin)，且第二目標係CD79。在一些實施例中，第二抗原結合部份係英妥珠單抗(inotuzumab)，藥物部份係奧唑米星(ozogamicin)，且第二目標係CD22。在一些實施例中，第二抗原結合部份係吉妥珠單抗(gemtuzumab)，藥物部份係奧唑米星(ozogamicin)，且第二目標係CD33。在一些實施例中，第二抗原結合部份係隆卡妥昔單抗(loncastuximab)，藥物部份係特西林(tesirine)，且第二目標係CD19。在一些實施例中，第二抗原結合部份係貝蘭單抗(belantamab)，藥物部份係莫福汀(mafodotin)，且第二目標係BCMA。在一些實施例中，第二抗原結合部份係米爾唯土西單抗(mirvetuximab)，藥物部份係索星(soravtansine)，且第二目標係FR⍺。在一些實施例中，第二抗原結合部份係莫昔土莫單抗(moxetumomab)，藥物部份係帕蘇多托克斯(pasudotox)，且第二目標係CD22。In some embodiments, the bispecific antibody provided herein comprises an FAP antigen-binding moiety and a second antigen-binding moiety that targets a second target and is attached to the drug moiety. In some embodiments, the second antigen-binding moiety is sacituzumab, the drug moiety is govitecan, and the second target is TROP2. In some embodiments, the second antigen-binding moiety is tisotumab, the drug moiety is vedotin, and the second target is tissue factor. In some embodiments, the second antigen-binding moiety is enfortumab, the drug moiety is vedotin, and the second target is Nectin4. In some embodiments, the secondary antigen-binding moiety is brentuximab, the drug portion is vedotin, and the secondary target is CD30. In some embodiments, the secondary antigen-binding moiety is trastuzumab, the drug portion is deruxtecan, and the secondary target is HER2. In some embodiments, the secondary antigen-binding moiety is trastuzumab, the drug portion is emtansine, and the secondary target is HER2. In some embodiments, the secondary antigen-binding moiety is polatuzumab, the drug portion is vedotin, and the secondary target is CD79. In some embodiments, the secondary antigen-binding moiety is inotuzumab, the drug portion is ozogamicin, and the secondary target is CD22. In some embodiments, the secondary antigen-binding moiety is gemtuzumab, the drug portion is ozogamicin, and the secondary target is CD33. In some embodiments, the secondary antigen-binding moiety is loncastuximab, the drug portion is tesirine, and the secondary target is CD19. In some embodiments, the secondary antigen-binding moiety is belantamab, the drug portion is mafodotin, and the secondary target is BCMA. In some embodiments, the second antigen-binding part is mirvetuximab, the drug part is soravtansine, and the secondary target is FR⍺. In some embodiments, the second antigen-binding part is moxetumomab, the drug part is pasudotox, and the secondary target is CD22.

在一些實施例中，本文所述之雙特異性抗體包含融合蛋白。 融合蛋白 In some embodiments, the bispecific antibodies described herein comprise fusion proteins. Fusion Proteins

本文提供一種融合蛋白或其用途。在一些實施例中，本揭露係關於包含在雙特異性抗體中的融合蛋白。在一些實施例中，本文所述之雙特異性抗體包含融合蛋白，該融合蛋白包含與一或多個額外結合部份及可選地一或多個額外部份融合的FAP抗原結合部份或其部分（例如包含CDRH1、CDRH2、及CDRH3之抗FAP VH）。在一些實施例中，額外結合部份係如本文所述之第二結合部份。在一些實施例中，第二結合部份可係靶向TACR的部份。在一些實施例中，第二結合部份可係細胞介素部份或靶向TACR的第二抗體部份。在一些實施例中，額外部份可係遮蔽/可裂解部份、可偵測/診斷劑、效應細胞或其一部分、異源蛋白質或其部份、藥物部份（諸如細胞裂解劑）、或連接子中之任一或多者。This document provides a fusion protein or its uses. In some embodiments, this disclosure relates to a fusion protein contained in a bispecific antibody. In some embodiments, the bispecific antibody described herein comprises a fusion protein comprising a FAP antigen-binding moiety or a portion thereof fused to one or more additional binding moieties and optionally one or more additional portions (e.g., anti-FAP VH comprising CDRH1, CDRH2, and CDRH3). In some embodiments, the additional binding moiety is a second binding moiety as described herein. In some embodiments, the second binding moiety may be a TACR-targeting portion. In some embodiments, the second binding moiety may be an interleukin portion or a second antibody portion targeting TACR. In some embodiments, the additional portion may be one or more of the following: a masking/cleavable portion, a detectable/diagnostic agent, an effector cell or a portion thereof, a heterologous protein or a portion thereof, a drug portion (such as a cell lysing agent), or a linker.

在一些實施例中，本文所述之雙特異性抗體包含融合蛋白，該融合蛋白包含與額外結合部份融合的FAP抗原結合部份或其部分（例如包含CDRH1、CDRH2、及CDRH3之抗FAP VH）。在一些實施例中，結合部份包括與目標分子直接交互作用並與其結合的分子（例如肽、多肽、或蛋白質）或分子錯合物（例如二或更多種肽、多肽、或蛋白質）的特定區或組分。該部份可包括促進對目標結合位點的識別及結合特異性的官能基或結構元件。在一些實施例中，本文所述之雙特異性抗體包含融合蛋白，該融合蛋白包含與連接子融合的FAP抗原結合部份或其部分（例如包含CDRH1、CDRH2、及CDRH3之抗FAP VH），該連接子與一或多個額外結合部份融合。在一些實施例中，本文所述之雙特異性抗體包含FAP抗原結合部份或其部分（例如包含CDRH1、CDRH2、及CDRH3之抗FAP VH），該FAP抗原結合部份或其部分與額外結合部份融合、與連接子融合、與串聯連接的額外結合部份融合。在一些實施例中，融合蛋白具有如圖2或圖6所顯示的形式D。In some embodiments, the bispecific antibodies described herein comprise a fusion protein containing an FAP antigen-binding moiety or a portion thereof fused to an additional binding moiety (e.g., anti-FAP VH comprising CDRH1, CDRH2, and CDRH3). In some embodiments, the binding moiety comprises a specific region or component of a molecule (e.g., a peptide, polypeptide, or protein) or a molecular complex (e.g., two or more peptides, polypeptides, or proteins) that directly interacts with and binds to the target molecule. This moiety may include functional groups or structural elements that facilitate recognition of the target binding site and binding specificity. In some embodiments, the bispecific antibody described herein comprises a fusion protein containing an FAP antigen-binding moiety or a portion thereof fused to a linker (e.g., anti-FAP VH comprising CDRH1, CDRH2, and CDRH3), the linker being fused to one or more additional binding moieties. In some embodiments, the bispecific antibody described herein comprises an FAP antigen-binding moiety or a portion thereof (e.g., anti-FAP VH comprising CDRH1, CDRH2, and CDRH3), the FAP antigen-binding moiety or a portion thereof being fused to additional binding moieties, fused to a linker, or fused to tandemly linked additional binding moieties. In some embodiments, the fusion protein has form D as shown in Figure 2 or Figure 6.

在一些實施例中，本揭露係關於融合蛋白，其包括FAP抗原結合部份或其部分（例如包含CDRH1、CDRH2、及CDRH3之抗FAP VH）及能夠刺激免疫細胞的細胞介素部份。在一些實施例中，本文所提供之融合蛋白係抗FAP細胞介素融合蛋白。In some embodiments, this disclosure relates to fusion proteins comprising an FAP antigen-binding portion or a portion thereof (e.g., an anti-FAP VH containing CDRH1, CDRH2, and CDRH3) and an intercytokine portion capable of stimulating immune cells. In some embodiments, the fusion protein provided herein is an anti-FAP intercytokine fusion protein.

在一些實施例中，FAP抗原結合部份包括上文所描述之抗FAP抗體或其抗原結合片段的部分或全長。In some embodiments, the FAP antigen-binding portion includes part or the full length of the anti-FAP antibody or its antigen-binding fragment as described above.

在一些實施例中，雙特異性抗體包含二或更多種融合蛋白。在一些實施例中，雙特異性抗體包含二或更多種融合蛋白，其中第一融合蛋白包含重鏈1 (HC1)區且第二融合蛋白包含重鏈2 (HC2)區。In some embodiments, the bispecific antibody comprises two or more fusion proteins. In some embodiments, the bispecific antibody comprises two or more fusion proteins, wherein the first fusion protein comprises a heavy chain 1 (HC1) region and the second fusion protein comprises a heavy chain 2 (HC2) region.

在一些實施例中，融合蛋白包含重鏈1 (HC1)區，其包含VH、重鏈恆定域1 (CH1)、及包含重鏈恆定域2 (CH2)及重鏈恆定域3 (CH3)之Fc片段。在一些實施例中，HC1區包含與如SEQ ID NO: 1、4、5、8、10、13、14、及15所示之胺基酸序列中之任一者包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列。在一些實施例中，HC1區包含與如SEQ ID NO: 5、8、10、13、14、及15所示之胺基酸序列中之任一者包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列。在一些實施例中，HC1區之Fc片段包含與Fc片段之C端融合的第二結合部份之一或多個單元。在一些實施例中，HC1區之Fc片段包含與HC1 Fc片段之C端融合的第二結合部份之第一單元。在一些實施例中，第二結合部份之第一單元係藉由第一連接子融合至Fc單元。在一些實施例中，第二結合部份係細胞介素部份，且其中細胞介素部份包含第一細胞介素單元。在一些實施例中，第一細胞介素單元包含LIGHT單元或淋巴毒素β單元。在一些實施例中，第一單元包含與表10所述之胺基酸序列中之任一者具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%同一性或與其同一之胺基酸序列。在一些實施例中，單元包含與SEQ ID NO: 17或SEQ ID NO: 39具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%同一性或與其同一之胺基酸序列。In some embodiments, the fusion protein includes a heavy chain 1 (HC1) region comprising VH, heavy chain constant 1 (CH1), and an Fc fragment comprising heavy chain constant 2 (CH2) and heavy chain constant 3 (CH3). In some embodiments, the HC1 region comprises an amino acid sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with or is identical to any of the amino acid sequences shown in SEQ ID NO: 1, 4, 5, 8, 10, 13, 14, and 15. In some embodiments, the HC1 region includes an amino acid sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with or identical to any of the amino acid sequences shown in SEQ ID NO: 5, 8, 10, 13, 14, and 15. In some embodiments, the Fc fragment of the HC1 region includes one or more units of a second binding portion fused to the C-terminus of the Fc fragment. In some embodiments, the Fc fragment of the HC1 region includes a first unit of a second binding portion fused to the C-terminus of the HC1 Fc fragment. In some embodiments, the first unit of the second binding portion is fused to the Fc unit via a first linker. In some embodiments, the second binding portion is an intercytokine portion, wherein the intercytokine portion includes a first intercytokine unit. In some embodiments, the first intercytokine unit comprises a LIGHT unit or a lymphotoxin β unit. In some embodiments, the first unit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with or identical to any of the amino acid sequences described in Table 10. In some embodiments, the unit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with or identical to SEQ ID NO: 17 or SEQ ID NO: 39.

在一些實施例中，融合蛋白包含重鏈2 (HC2)區，其包含VH、CH1、及包含CH1及CH3之Fc片段。在一些實施例中，HC2區包含與如SEQ ID NO: 1、4、6、9、11、12、13、及16所示之胺基酸序列中之任一者包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列。在一些實施例中，HC2區包含與如SEQ ID NO: 6、9、11、12、13、及16所示之胺基酸序列中之任一者包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列。在一些實施例中，HC2區之Fc片段包含與HC2 Fc片段之C端融合的第二結合部份之一或多個單元。在一些實施例中，HC2區之Fc片段包含第二結合部份之第二單元及第三單元。在一些實施例中，第二結合部份之第二單元係融合至HC2區之Fc片段。在一些實施例中，第二結合部份之第三單元係融合至第二結合部份之第二單元。在一些實施例中，第二結合部份係藉由第二連接子融合至HC2區之Fc片段，且第三單元係藉由第三連接子融合至第二結合部份之第二單元。在一些實施例中，第二結合部份單元之第二單元及第三單元係串聯連接。在一些實施例中，第二結合部份係細胞介素部份，且其中細胞介素部份包含第二細胞介素單元及第三細胞介素單元。在一些實施例中，第二細胞介素單元包含LIGHT單元、淋巴毒素α單元、或淋巴毒素β單元。在一些實施例中，第三細胞介素單元包含LIGHT單元、淋巴毒素α單元、或淋巴毒素β單元。在一些實施例中，第二細胞介素單元包含LIGHT單元，且第三細胞介素單元包含LIGHT單元。在一些實施例中，第二細胞介素單元包含淋巴毒素α單元，且第三細胞介素單元包含淋巴毒素β單元。在一些實施例中，第二單元包含與表10所述之胺基酸序列中之任一者具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%同一性或與其同一之胺基酸序列。在一些實施例中，第二單元包含與SEQ ID NO: 17或SEQ ID NO: 40具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%同一性或與其同一之胺基酸序列。在一些實施例中，第三單元包含與表10所述之胺基酸序列中之任一者具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%同一性或與其同一之胺基酸序列。在一些實施例中，第三單元包含與SEQ ID NO: 17或SEQ ID NO: 39具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%同一性或與其同一之胺基酸序列。In some embodiments, the fusion protein includes a heavy chain 2 (HC2) region comprising VH, CH1, and an Fc fragment comprising CH1 and CH3. In some embodiments, the HC2 region comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identical or identical to any of the amino acid sequences shown in SEQ ID NO: 1, 4, 6, 9, 11, 12, 13, and 16. In some embodiments, the HC2 region comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identical or identical to any of the amino acid sequences shown in SEQ ID NO: 6, 9, 11, 12, 13, and 16. In some embodiments, the Fc segment of the HC2 region includes one or more units of a second bonding portion fused to the C-terminus of the HC2 Fc segment. In some embodiments, the Fc segment of the HC2 region includes a second unit and a third unit of the second bonding portion. In some embodiments, the second unit of the second bonding portion is fused to the Fc segment of the HC2 region. In some embodiments, the third unit of the second bonding portion is fused to the second unit of the second bonding portion. In some embodiments, the second bonding portion is fused to the Fc segment of the HC2 region via a second connector, and the third unit is fused to the second unit of the second bonding portion via a third connector. In some embodiments, the second and third units of the second bonding portion unit are connected in series. In some embodiments, the second binding portion is an interleukin portion, wherein the interleukin portion includes a second interleukin unit and a third interleukin unit. In some embodiments, the second interleukin unit includes a LIGHT unit, a lymphotoxin alpha unit, or a lymphotoxin beta unit. In some embodiments, the third interleukin unit includes a LIGHT unit, a lymphotoxin alpha unit, or a lymphotoxin beta unit. In some embodiments, the second interleukin unit includes a LIGHT unit, and the third interleukin unit includes a LIGHT unit. In some embodiments, the second interleukin unit includes a lymphotoxin alpha unit, and the third interleukin unit includes a lymphotoxin beta unit. In some embodiments, the second unit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with or the same as any of the amino acid sequences described in Table 10. In some embodiments, the second unit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with or the same as any of the amino acid sequences described in Table 10. In some embodiments, the third unit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with or the same as any of the amino acid sequences described in Table 10. In some embodiments, the third unit comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to or the same as SEQ ID NO: 17 or SEQ ID NO: 39.

在一些實施例中，融合蛋白包含一或多個連接子。本文進一步描述連接子。In some embodiments, the fusion protein includes one or more linkers. Linkers are further described herein.

在一些實施例中，融合蛋白包含一或多個異二聚化修飾。在一些實施例中，HC1區包含一或多個異二聚化修飾。在一些實施例中，HC1區之一或多個異二聚化修飾係鈕修飾或孔修飾。在一些實施例中，HC1區之一或多個異二聚化修飾係孔修飾。在一些實施例中，HC2包含一或多個異二聚化修飾。在一些實施例中，HC2區之一或多個異二聚化修飾係鈕修飾或孔修飾。在一些實施例中，HC2區之一或多個異二聚化修飾係鈕修飾。 例示性抗FAP抗原結合部份 In some embodiments, the fusion protein includes one or more heterodimerization modifications. In some embodiments, the HC1 region includes one or more heterodimerization modifications. In some embodiments, one or more heterodimerization modifications of the HC1 region are button modifications or pore modifications. In some embodiments, one or more heterodimerization modifications of the HC1 region are pore modifications. In some embodiments, the HC2 region includes one or more heterodimerization modifications. In some embodiments, one or more heterodimerization modifications of the HC2 region are button modifications or pore modifications. In some embodiments, one or more heterodimerization modifications of the HC2 region are button modifications. Exemplary anti-FAP antigen binding region

在一些實施例中，FAP抗原結合部份包括具有如KTNQNVDYX 1GNTFMH (SEQ ID NO: 23)所示之胺基酸序列的LCDR1（其中X 1係N或S）、具有如LASNLAS (SEQ ID NO: 24)所示之胺基酸序列的LCDR2、具有如QQSRNLPYT (SEQ ID NO: 25)所示之胺基酸序列的LCDR3、具有如IYGVN (SEQ ID NO: 26)所示之胺基酸序列的HCDR1、具有如AIWSGGRKDYX 2LSLKS (SEQ ID NO: 27)所示之胺基酸序列的HCDR2（其中X 2係N或S）、及具有如SQDMPGYFDY (SEQ ID NO: 28)所示之胺基酸序列的HCDR3。 In some embodiments, the FAP antigen binding portion includes LCDR1 (where X1 is N or S) having an amino acid sequence as shown in KTNQNVDYX 1 GNTFMH (SEQ ID NO: 23), LCDR2 having an amino acid sequence as shown in LASSNLAS (SEQ ID NO: 24), LCDR3 having an amino acid sequence as shown in QQSRNLPYT (SEQ ID NO: 25), HCDR1 having an amino acid sequence as shown in IYGVN (SEQ ID NO: 26), HCDR2 (where X2 is N or S) having an amino acid sequence as shown in AIWSGGRKDYX 2 LSLKS (SEQ ID NO: 27), and HCDR3 having an amino acid sequence as shown in SQDMPGYFDY (SEQ ID NO: 28).

在一些實施例中，FAP抗原結合部份包括具有如SASSRVGYMH (SEQ ID NO: 29)所示之胺基酸序列的LCDR1、具有如DTSKLAS (SEQ ID NO: 30)所示之胺基酸序列的LCDR2、具有如FQGSGYPFT (SEQ ID NO: 31)所示之胺基酸序列的LCDR3、具有如TAGMSVG (SEQ ID NO: 32)所示之胺基酸序列的HCDR1、具有如DIWWDDKKHYNPSLKD (SEQ ID NO: 33)所示之胺基酸序列的HCDR2、及具有如DMIFNFYFDV (SEQ ID NO: 34)所示之胺基酸序列的HCDR3。In some embodiments, the FAP antigen binding portion includes LCDR1 having an amino acid sequence as shown in SASSRVGYMH (SEQ ID NO: 29), LCDR2 having an amino acid sequence as shown in DTSKLAS (SEQ ID NO: 30), LCDR3 having an amino acid sequence as shown in FQGSGYPFT (SEQ ID NO: 31), HCDR1 having an amino acid sequence as shown in TAGMSVG (SEQ ID NO: 32), HCDR2 having an amino acid sequence as shown in DIWWDDKKHYNPSLKD (SEQ ID NO: 33), and HCDR3 having an amino acid sequence as shown in DMIFNFYFDV (SEQ ID NO: 34).

在一些實施例中，FAP抗原結合部份包括與上文所描述之抗FAP抗體或其抗原結合片段的VH及/或VL相同的VH及/或VL。In some embodiments, the FAP antigen-binding portion includes the same VH and/or VL as the anti-FAP antibody or its antigen-binding fragment described above.

在一些實施例中，融合蛋白包括第一重鏈、第二重鏈、及分別與第一重鏈及第二重鏈配對的兩條輕鏈，其中：(1)第一重鏈包含與如SEQ ID NO: 3所示之胺基酸序列具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、100%同一性之胺基酸序列；第二重鏈包含與如SEQ ID NO: 5所示之胺基酸序列具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、100%同一性之胺基酸序列；輕鏈包含與如SEQ ID NO: 6所示之胺基酸序列具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、100%同一性之胺基酸序列；(2)第一重鏈包含與如SEQ ID NO: 7所示之胺基酸序列具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、100%同一性之胺基酸序列；第二重鏈包含與如SEQ ID NO: 8所示之胺基酸序列具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、100%同一性之胺基酸序列；輕鏈包含與如SEQ ID NO: 9所示之胺基酸序列具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、100%同一性之胺基酸序列；(3)第一重鏈包含與如SEQ ID NO: 3所示之胺基酸序列具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、100%同一性之胺基酸序列；第二重鏈包含與如SEQ ID NO: 10所示之胺基酸序列具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、100%同一性之胺基酸序列；輕鏈包含與如SEQ ID NO: 11所示之胺基酸序列具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、100%同一性之胺基酸序列；(4)第一重鏈包含與如SEQ ID NO: 3所示之胺基酸序列具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、100%同一性之胺基酸序列；第二重鏈包含與如SEQ ID NO: 15所示之胺基酸序列具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、100%同一性之胺基酸序列；輕鏈包含與如SEQ ID NO: 16所示之胺基酸序列具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、100%同一性之胺基酸序列。In some embodiments, the fusion protein includes a first heavy chain, a second heavy chain, and two light chains respectively paired with the first heavy chain and the second heavy chain, wherein: (1) the first heavy chain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 3; the second heavy chain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 5; the light chains comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 5; The amino acid sequence shown in SEQ ID NO: 6 has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% identity with the amino acid sequence shown in SEQ ID NO: 7; (2) the first heavy chain contains an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% identity with the amino acid sequence shown in SEQ ID NO: 8; the second heavy chain contains an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% identity with the amino acid sequence shown in SEQ ID NO: 8; the light chain contains an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% identity with the amino acid sequence shown in SEQ ID NO: 7; The amino acid sequence shown in SEQ ID NO: 9 has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% identity with the amino acid sequence shown in SEQ ID NO: 3; (3) the first heavy chain contains an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% identity with the amino acid sequence shown in SEQ ID NO: 3; the second heavy chain contains an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% identity with the amino acid sequence shown in SEQ ID NO: 10; the light chain contains an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% identity with the amino acid sequence shown in SEQ ID NO: 10; The amino acid sequence shown in SEQ ID NO: 11 has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% identity with the amino acid sequence shown in SEQ ID NO: 3; (4) the first heavy chain contains an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% identity with the amino acid sequence shown in SEQ ID NO: 3; the second heavy chain contains an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% identity with the amino acid sequence shown in SEQ ID NO: 15; the light chain contains an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% identity with the amino acid sequence shown in SEQ ID NO: 15; The amino acid sequences shown in 16 have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% identity.

在一些實施例中，融合蛋白包括具有如SEQ ID NO: 3所示之胺基酸序列的第一重鏈、具有如SEQ ID NO: 5所示之胺基酸序列的第二重鏈、及具有如SEQ ID NO: 6所示之胺基酸序列的輕鏈。在一些實施例中，融合蛋白包括具有如SEQ ID NO: 7所示之胺基酸序列的第一重鏈、具有如SEQ ID NO: 8所示之胺基酸序列的第二重鏈、及具有如SEQ ID NO: 9所示之胺基酸序列的輕鏈。在一些實施例中，融合蛋白包括具有如SEQ ID NO: 3所示之胺基酸序列的第一重鏈、具有如SEQ ID NO: 10所示之胺基酸序列的第二重鏈、及具有如SEQ ID NO: 11所示之胺基酸序列的輕鏈。在一些實施例中，融合蛋白包括具有如SEQ ID NO: 3所示之胺基酸序列的第一重鏈、具有如SEQ ID NO: 15所示之胺基酸序列的第二重鏈、及具有如SEQ ID NO: 16所示之胺基酸序列的輕鏈。In some embodiments, the fusion protein includes a first heavy chain having an amino acid sequence as shown in SEQ ID NO: 3, a second heavy chain having an amino acid sequence as shown in SEQ ID NO: 5, and a light chain having an amino acid sequence as shown in SEQ ID NO: 6. In some embodiments, the fusion protein includes a first heavy chain having an amino acid sequence as shown in SEQ ID NO: 7, a second heavy chain having an amino acid sequence as shown in SEQ ID NO: 8, and a light chain having an amino acid sequence as shown in SEQ ID NO: 9. In some embodiments, the fusion protein includes a first heavy chain having an amino acid sequence as shown in SEQ ID NO: 3, a second heavy chain having an amino acid sequence as shown in SEQ ID NO: 10, and a light chain having an amino acid sequence as shown in SEQ ID NO: 11. In some embodiments, the fusion protein includes a first heavy chain having an amino acid sequence as shown in SEQ ID NO: 3, a second heavy chain having an amino acid sequence as shown in SEQ ID NO: 15, and a light chain having an amino acid sequence as shown in SEQ ID NO: 16.

FAP抗原結合部份之另外CDR、VH、VL、HC、及/或LC在本文的FAP結合劑部分中詳細描述。The other CDR, VH, VL, HC, and/or LC of the FAP antigen-binding portion are described in detail in the FAP binder section of this document.

在一些實施例中，融合蛋白在FAP抗原結合部份之N端處包括衍生自免疫球蛋白之Fc片段。在一些實施例中，Fc片段係衍生自IgG1、IgG2、IgG3、或IgG4，可選地，Fc片段係衍生自IgG4。在一些實施例中，Fc片段包括突變S228P。在一些實施例中，Fc片段包括LALAPG突變。在一些實施例中，Fc片段包括選自由下列所組成之群組的一或多個修飾：鈕扣、DDKK、CH3之靜電轉向、DuoBody、SEEDbodies、cFAE、XmAb、Azymetric、及BEAT ®。在一些實施例中，Fc片段包括修飾鈕扣及/或DDKK。 In some embodiments, the fusion protein includes an immunoglobulin-derived Fc fragment at the N-terminus of the FAP antigen-binding site. In some embodiments, the Fc fragment is derived from IgG1, IgG2, IgG3, or IgG4; alternatively, the Fc fragment is derived from IgG4. In some embodiments, the Fc fragment includes the S228P mutation. In some embodiments, the Fc fragment includes the LALAPG mutation. In some embodiments, the Fc fragment includes one or more modifications selected from the group consisting of: button, DDKK, electrostatic switching of CH3, DuoBody, SEEDbodies, cFAE, XmAb, Azymetric, and BEAT® . In some embodiments, the Fc fragment includes modified button and/or DDKK.

在本揭露中，融合蛋白可包括經保守修飾的變體，諸如FR區或Fc區。經保守修飾的變體包括多肽序列的個別取代、缺失、或添加，這導致胺基酸經化學上相似的胺基酸取代。提供功能相似胺基酸的保守性取代表係所屬技術領域中熟知的且在本文進一步描述。本文進一步描述其他胺基酸修飾。 例示性細胞介素部份 In this disclosure, fusion proteins may include conserved variants, such as FR or Fc regions. Conserved variants include individual substitutions, deletions, or additions of the polypeptide sequence, resulting in the substitution of an amino acid with a chemically similar amino acid. Conserved substitutions of functionally similar amino acids are well known in the art and are further described herein. Other amino acid modifications are further described herein. Exemplary Intercytokine Section

例示性細胞介素部份在本文中詳細描述，例如在雙特異性抗體部分。細胞介素部份可在融合蛋白之C端。在一些實施例中，細胞介素部份係可操作地連接至Fc片段之C端。在一些實施例中，細胞介素部份直接連接至Fc片段。在一些實施例中，細胞介素部份經由連接子連接至Fc片段。在一些實施例中，細胞介素部份包含第一細胞介素部份及第二細胞介素部份，第一細胞介素部份包含一個細胞介素突變體且第二細胞介素部份包含串聯連接的兩個細胞介素突變體，第一細胞介素部份及第二細胞介素部份連接至Fc片段內之蛋白質片段的不同C端。在一些實施例中，第一細胞介素部份連接至具有孔突變之Fc片段之C端，且第二細胞介素部份連接至具有鈕突變之Fc片段之C端。Exemplary intercytokine moieties are described in detail herein, such as in bispecific antibody moieties. The intercytokine moiety may be located at the C-terminus of the fusion protein. In some embodiments, the intercytokine moiety is operatively linked to the C-terminus of the Fc fragment. In some embodiments, the intercytokine moiety is directly linked to the Fc fragment. In some embodiments, the intercytokine moiety is linked to the Fc fragment via a linker. In some embodiments, the intercytokine portion includes a first intercytokine portion and a second intercytokine portion, the first intercytokine portion including one intercytokine mutant and the second intercytokine portion including two intercytokine mutants connected in tandem, the first and second intercytokine portions being linked to different C-termini within the Fc fragment. In some embodiments, the first intercytokine portion is linked to the C-terminus of an Fc fragment with a pore mutation, and the second intercytokine portion is linked to the C-terminus of an Fc fragment with a button mutation.

在一些實施例中，細胞介素突變體係LIGHT突變體，第一細胞介素部份含有一個LIGHT突變體，且第二細胞介素部份含有串聯連接的兩個LIGHT突變體。在一些實施例中，第一細胞介素部份包含一個淋巴毒素-β突變體，且第二細胞介素部份包含串聯連接的淋巴毒素-αβ突變體，淋巴毒素-β突變體包括如SEQ ID NO: 39所示之胺基酸序列，淋巴毒素-α突變體包含如SEQ ID NO: 40所示之胺基酸序列。在一些實施例中，第一細胞介素部份經由連接子A連接至Fc片段，且第二細胞介素部份經由連接子B連接至Fc片段。在一些實施例中，串聯連接的兩個LIGHT突變體直接或經由連接子C彼此連接。在一些實施例中，LIGHT變體包括如SEQ ID NO: 17或18所示的胺基酸序列。In some embodiments, the interleukin mutant is a LIGHT mutant, with the first interleukin portion containing one LIGHT mutant and the second interleukin portion containing two LIGHT mutants linked in series. In some embodiments, the first interleukin portion includes a lymphotoxin-β mutant and the second interleukin portion includes lymphotoxin-αβ mutants linked in series, the lymphotoxin-β mutant including the amino acid sequence shown in SEQ ID NO: 39 and the lymphotoxin-α mutant including the amino acid sequence shown in SEQ ID NO: 40. In some embodiments, the first interleukin portion is linked to the Fc fragment via linker A, and the second interleukin portion is linked to the Fc fragment via linker B. In some embodiments, the two LIGHT mutants are connected to each other directly or via connector C. In some embodiments, the LIGHT variants include an amino acid sequence as shown in SEQ ID NO: 17 or 18.

在一些實施例中，融合蛋白以不大於20 nM、15 nM、10 nM、或5 nM之解離常數（KD，KD=koff/kon，或KD= Kd/Ka）結合至FAP。在一些實施例中，融合蛋白以不大於20 nM、15 nM、10 nM、或5 nM之解離常數(KD)結合至LTßR。在一些實施例中，融合蛋白幾乎不結合至人類或食蟹獼猴HVEM。在一些實施例中，LIGHT突變體能夠降低與DcR3之結合親和力。在一些實施例中，融合蛋白特異性結合至人類FAP，且/或不結合至DPPIV。本文進一步描述融合蛋白之活性。In some embodiments, the fusion protein binds to FAP with a dissociation constant (KD, KD = koff/kon, or KD = Kd/Ka) not greater than 20 nM, 15 nM, 10 nM, or 5 nM. In some embodiments, the fusion protein binds to LTßR with a dissociation constant (KD) not greater than 20 nM, 15 nM, 10 nM, or 5 nM. In some embodiments, the fusion protein hardly binds to human or cynomolgus monkey HVEM. In some embodiments, the LIGHT mutant is able to reduce the binding affinity to DcR3. In some embodiments, the fusion protein specifically binds to human FAP and/or does not bind to DPPIV. The activity of the fusion protein is further described herein.

使用平台D產生的融合蛋白能夠穩健地產生免疫細胞介素，顯示出有前景的抗腫瘤功效。 遮蔽/可裂解部份 The fusion protein produced using Platform D robustly generates immunocytokines, demonstrating promising antitumor efficacy. Catalyzable/Cleavable Parts

在一些實施例中，融合蛋白之FAP結合部分（例如抗FAP抗體、FAP抗原結合部份、或包含FAP抗原結合部份之雙特異性抗體）與遮蔽部份及/或可裂解部份（直接或間接）連接、融合、或接合，其中融合蛋白之FAP結合部分（例如抗FAP抗體、FAP抗原結合部份、或包含FAP抗原結合部份之雙特異性抗體）的一或多個FAP結合部份經遮蔽（例如，經由遮蔽部份）及/或係可活化的（例如，經由可裂解部份）。用於遮蔽融合蛋白的FAP結合部分（例如抗FAP抗體、FAP抗原結合部份、或包含FAP抗原結合部份之雙特異性抗體）的技術係所屬技術領域中熟知的，包括SAFE body遮蔽技術（參見例如美國專利申請公開案第2019/0241886號）及Probody遮蔽技術（參見例如美國專利申請公開案第2015/0079088號）。此類技術可用以產生經遮蔽及/或可活化的融合蛋白之FAP結合部分（例如抗FAP抗體、FAP抗原結合部份、或包含FAP抗原結合部份之雙特異性抗體）。融合蛋白的此類經遮蔽的及/或可活化的FAP結合部分（例如抗FAP抗體、FAP抗原結合部份、或包含FAP抗原結合部份之雙特異性抗體）可用於製備接合物，包括免疫接合物、抗體藥物接合物(antibody-drug conjugate, ADC)、經遮蔽的ADC、及可活化的抗體藥物接合物(activatable antibody-drug conjugate, AADC)，該等接合物包含本揭露之融合蛋白的FAP結合部分（例如抗FAP抗體、FAP抗原結合部份、或包含FAP抗原結合部份之雙特異性抗體）中之任一者，諸如融合蛋白的人類FAP結合部分，包括直接或間接連接至具有效應功能之第二試劑者。In some embodiments, the FAP-binding portion of the fusion protein (e.g., an anti-FAP antibody, an FAP antigen-binding portion, or a bispecific antibody containing an FAP antigen-binding portion) is (directly or indirectly) linked, fused, or conjugated to a masking portion and/or a cleavable portion, wherein one or more FAP-binding portions of the fusion protein (e.g., an anti-FAP antibody, an FAP antigen-binding portion, or a bispecific antibody containing an FAP antigen-binding portion) are masked (e.g., via a masking portion) and/or activated (e.g., via a cleavable portion). Techniques for masking the FAP-binding portion of a fusion protein (e.g., anti-FAP antibodies, FAP antigen-binding portions, or bispecific antibodies containing FAP antigen-binding portions) are well known in the art, including SAFE body masking techniques (see, for example, U.S. Patent Application Publication No. 2019/0241886) and Probody masking techniques (see, for example, U.S. Patent Application Publication No. 2015/0079088). Such techniques can be used to generate masked and/or activated FAP-binding portions of fusion proteins (e.g., anti-FAP antibodies, FAP antigen-binding portions, or bispecific antibodies containing FAP antigen-binding portions). Such masked and/or activatable FAP-binding portions of fusion proteins (e.g., anti-FAP antibodies, FAP antigen-binding portions, or bispecific antibodies containing FAP antigen-binding portions) can be used to prepare conjugates, including immunoconjugates, antibody-drug conjugates (ADCs), masked ADCs, and activatable antibody-drug conjugates (AADCs), which contain any of the FAP-binding portions of the fusion proteins disclosed herein (e.g., anti-FAP antibodies, FAP antigen-binding portions, or bispecific antibodies containing FAP antigen-binding portions), such as the human FAP-binding portion of the fusion protein, including those directly or indirectly linked to a second reagent with efficacy.

在一些實施例中，融合蛋白的FAP結合部分與第二試劑（直接或間接）連接、融合、或接合。在一些實施例中，第二試劑包含診斷劑、可偵測劑、或治療劑，諸如細胞裂解劑、效應細胞、或異源蛋白質。 可偵測/診斷劑 In some embodiments, the FAP-binding portion of the fusion protein is linked, fused, or conjugated (directly or indirectly) to a second reagent. In some embodiments, the second reagent comprises a diagnostic agent, a detectable agent, or a therapeutic agent, such as a cell lysing agent, effector cells, or a heterologous protein. Detectable/Diagnostic Agent

在一些實施例中，本文所提供之融合蛋白之FAP結合部分可與可偵測劑或診斷劑連接、融合、或接合。可偵測劑的實例包括各種酶、輔基、螢光材料、發光材料、生物發光材料、放射性材料、正電子發射金屬、非放射性順磁性金屬離子、及反應性部份。可偵測劑可使用所屬技術領域中已知的技術直接或間接地耦合或接合至抗體或其片段，例如透過所屬技術領域中已知的連接子或另一部份。酶標記之實例包括螢光素酶（例如螢火蟲螢光素酶及細菌螢光素酶；美國專利第4,737,456號）、螢光素、2,3-二氫酞嗪二酮類、蘋果酸去氫酶、脲酶、過氧化酶（諸如辣根過氧化酶(horseradish peroxidase, HRPO)）、鹼性磷酸酶、β-半乳糖苷酶、乙醯膽鹼酯酶、葡糖澱粉酶、溶菌酶、糖氧化酶（例如葡萄糖氧化酶、半乳糖氧化酶、及葡萄糖-6-磷酸去氫酶）、雜環氧化酶（如尿酸酶及黃嘌呤氧化酶）、乳過氧化酶、微過氧化酶、及類似者。In some embodiments, the FAP-binding portion of the fusion protein provided herein may be linked, fused, or conjugated to a detectable or diagnostic agent. Examples of detectable agents include various enzymes, cofactors, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron-emitting metals, non-radioactive paramagnetic metal ions, and reactive portions. Detectable agents may be directly or indirectly coupled or conjugated to antibodies or fragments thereof using techniques known in the art, such as through linkers or other portions known in the art. Examples of enzyme-labeled enzymes include luciferases (e.g., firefly luciferase and bacterial luciferase; U.S. Patent No. 4,737,456), luciferin, 2,3-dihydrophthalimide diones, malic acid dehydrogenase, urease, peroxidases (such as horseradish peroxidase (HRPO)), alkaline phosphatase, β-galactosidase, acetylcholinesterase, glucose amylase, lysozyme, sugar oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and similar enzymes.

適合的輔基錯合物的實例包括鏈黴抗生物素/生物素及抗生物素蛋白/生物素；適合的螢光材料的實例包括繖形酮(umbelliferone)、螢光素、螢光異硫氰酸鹽、玫瑰紅(rhodamine)、二氯三嗪胺螢光素、丹磺醯氯、或藻紅素；發光材料的實例包括魯米諾(luminol)；生物發光材料的實例包括螢光素酶、螢光素、及水母發光蛋白；且適合的放射性材料的實例包括125I、131I、111In、或99mTc。Examples of suitable coenzyme complexes include streptavidin/biotin and avidin/biotin; examples of suitable luminescent materials include umbelliferone, luminescent pigment, luminescent isothiocyanate, rhodamine, dichlorotriazineamine luminescent pigment, dansyl chloride, or phycoerythrin; examples of luminescent materials include luminol; examples of bioluminescent materials include luminescent enzyme, luminescent pigment, and jellyfish luminescent protein; and examples of suitable radioactive materials include 125I, 131I, 111In, or 99mTc.

FAP表現的偵測通常涉及將生物樣本（個體的腫瘤、細胞、組織、或體液）與本文所提供之融合蛋白的一或多個FAP結合部分（可選地接合至可偵測部份）接觸，且偵測樣本是否呈現FAP陽性，或樣本與對照樣本相比表現是否改變（例如降低或增加）。本文進一步描述其他可偵測劑及診斷劑，例如作為可偵測標記及/或診斷標記。 效應細胞 Detection of FAP performance typically involves contacting a biological sample (an individual's tumor, cells, tissue, or body fluid) with one or more FAP-binding portions of the fusion protein described herein (optionally binding to a detectable portion), and determining whether the sample is FAP-positive or whether its performance has changed (e.g., decreased or increased) compared to a control sample. This document further describes other detectable and diagnostic agents, for example, as detectable and/or diagnostic markers. Effective Cells

在一些實施例中，本文所提供之融合蛋白的FAP結合部分與效應細胞連接、接合、或融合。在一些實施例中，效應細胞包含本文所述之免疫細胞（例如NK細胞、樹突、B細胞、巨噬細胞、及類似者）或表現嵌合抗原受體之T細胞（CAR T細胞），其經工程改造用於趨化因子受體或FAP靶向。最近，CAR T細胞因其臨床成功而受到關注，且加快了FDA的批准，請參閱WO2020102240，該文獻全部併入本文中。在CAR T細胞方法中，從患者血液中收集T細胞，且接著進行基因工程改造，以表現對腫瘤細胞上存在的抗原具有特異性的CAR。接著向同一患者重新投予此等經工程改造T細胞。一旦注射，CAR T細胞識別目標細胞上的標靶抗原，誘導目標細胞死亡。因此，CAR T細胞構成了諸如腫瘤治療的醫療用途的新模態。嵌合抗原受體(chimeric antigen receptor, CAR)係一種經基因工程改造受體，其經設計以靶向特定抗原，例如腫瘤抗原。例如，該靶向可導致針對腫瘤的細胞毒性，使得表現CAR之CAR T細胞可經由特定的腫瘤抗原靶向並殺滅腫瘤。根據本揭露，如本文所述之提供用於FAP識別之融合蛋白的FAP結合部分可用以工程改造CAR T細胞以特異性識別FAP表現性細胞。本文涵蓋的CAR可包含a)識別區，例如衍生自所提供之抗FAP或抗趨化因子受體抗體的單鏈片段可變(scFv)區，用於識別及結合至由目標細胞表現的FAP；及b)活化信號傳導域，例如T細胞的CD3鏈，其可用作CAR中的T細胞活化信號。In some embodiments, the FAP-binding portion of the fusion protein provided herein is linked, conjugated, or fused with effector cells. In some embodiments, the effector cells comprise immune cells described herein (e.g., NK cells, dendritic cells, B cells, macrophages, and similar cells) or T cells expressing chimeric antigen receptors (CAR T cells) engineered for chemokine receptor or FAP targeting. Recently, CAR T cells have gained attention due to their clinical success and accelerated FDA approval; see WO2020102240, which is incorporated herein in its entirety. In the CAR T cell approach, T cells are collected from a patient's blood and then genetically engineered to express a CAR specific for antigens present on tumor cells. These engineered T cells are then re-injected into the same patient. Once injected, the CAR T cells recognize the target antigen on the target cells, inducing the death of the target cells. Therefore, CAR T cells constitute a new paradigm for medical applications such as cancer treatment. A chimeric antigen receptor (CAR) is a genetically engineered receptor designed to target specific antigens, such as tumor antigens. For example, this targeting can lead to tumor-specific cytotoxicity, allowing CAR T cells expressing CARs to target and kill tumors via specific tumor antigens. According to this disclosure, the FAP-binding portion of the fusion protein provided herein for FAP recognition can be used to engineer CAR T cells to specifically recognize FAP-expressing cells. The CARs covered in this article may include a) a recognition region, such as a single-strand fragment variable (scFv) region derived from the provided anti-FAP or anti-chemofacilitator receptor antibody, for recognizing and binding to FAP expressed by the target cell; and b) an activation signaling domain, such as the CD3 chain of T cells, which can be used as a T cell activation signal in the CAR.

在一些實施例中，本文所提供之CAR包含共刺激域（例如CD137、CD28、或CD134）以在體內實現T細胞的延長活化。共刺激域的添加增強了含有CAR的T細胞的體內增殖及存活，且初始臨床數據已顯示此類構築體在治療疾病（諸如癌症）中係有前景的治療劑。在一些實施例中，本文所提供之CAR T細胞可用於本文所述之方法中。例如，此類CAR T細胞可用以治療具有局部或全身異常存在的表現FAP之細胞（諸如腫瘤相關T細胞）的任何疾病。 異源蛋白質 In some embodiments, the CARs provided herein include co-stimulatory domains (e.g., CD137, CD28, or CD134) to achieve T cell prolongation and activation in vivo. The addition of co-stimulatory domains enhances the in vivo proliferation and survival of CAR-containing T cells, and initial clinical data have shown that such constructs are promising therapeutic agents for diseases such as cancer. In some embodiments, the CAR T cells provided herein can be used in the methods described herein. For example, such CAR T cells can be used to treat any disease with FAP-like cells (such as tumor-associated T cells) exhibiting local or systemic abnormalities. Heterologous Proteins

在一些實施例中，本文所提供之融合蛋白的FAP結合部分與異源蛋白質或多肽（或其片段，例如與（例如約10、約20、約30、約40、約50、約60、約70、約80、約90、或約100個胺基酸的）多肽連接、接合、或融合。在一些實施例中，異源蛋白質與本揭露之融合蛋白的FAP結合部分的連接、接合、及/或融合產生融合蛋白，因此，本文提供融合蛋白以及其用途。在一些實施例中，本文描述融合蛋白，其包含融合蛋白之FAP結合部分之抗原結合片段（例如抗FAP抗體、FAP抗原結合部份、或包含FAP抗原結合部份之雙特異性抗體），包括本文描述的人類FAP結合劑（例如包含VH及/或VL之CDR1、CDR2、及/或CDR3）及異源蛋白質、多肽、或肽。在一些實施例中，與融合蛋白之FAP結合部分（例如抗FAP抗體、FAP抗原結合部份、或包含FAP抗原結合部份之雙特異性抗體）連接的異源蛋白質、多肽、或肽可用於將FAP結合劑靶向特定細胞（例如表現FAP之細胞，包括腫瘤細胞）。在一些實施例中，異源蛋白質係信號肽。在一些實施例中，異源蛋白質係細胞穿透肽。在一些實施例中，異源蛋白質係次細胞定位信號。 細胞裂解劑 In some embodiments, the FAP-binding portion of the fusion protein provided herein is linked, conjugated, or fused with a heterologous protein or polypeptide (or fragment thereof, such as with a polypeptide of about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 100 amino acids). In some embodiments, the linking, conjugation, and/or fusion of a heterologous protein with the FAP-binding portion of the fusion protein disclosed herein produces a fusion protein; therefore, fusion proteins and their uses are provided herein. In some embodiments, fusion proteins are described herein that include an antigen-binding fragment (e.g., an anti-FAP antibody, an FAP antigen-binding portion, or a fragment containing an FAP antigen-binding portion) of the fusion protein. Bispecific antibodies, including human FAP conjugates described herein (e.g., CDR1, CDR2, and/or CDR3 comprising VH and/or VL) and heterologous proteins, peptides, or peptides. In some embodiments, the heterologous protein, peptide, or peptide linked to the FAP-binding portion of the fusion protein (e.g., an anti-FAP antibody, an FAP antigen-binding portion, or a bispecific antibody comprising an FAP antigen-binding portion) can be used to target the FAP conjugate to specific cells (e.g., cells expressing FAP, including tumor cells). In some embodiments, the heterologous protein is a signaling peptide. In some embodiments, the heterologous protein is a cell-penetrating peptide. In some embodiments, the heterologous protein is a subcellular localization signal. Cell lysing agents

在一些實施例中，本文所提供之融合蛋白之FAP結合部分係與一或多種細胞裂解劑連接、接合、或融合。如本文中所使用，細胞裂解劑係降低一或多種細胞之增殖能力的部份。當細胞變得不太能夠增殖時，例如，因為細胞經歷細胞凋亡或以其他方式死亡，細胞不能進行細胞週期且/或不能分裂，細胞分化等，細胞具有降低的增殖能力。非限制性例示性細胞裂解劑包括但不限於放射性同位素、光敏劑(photosensitizer, PS)、細胞毒素、及化學治療劑。In some embodiments, the FAP-binding portion of the fusion protein provided herein is linked, bound, or fused with one or more cytolytic agents. As used herein, a cytolytic agent is a portion that reduces the proliferative capacity of one or more cells. Cells have reduced proliferative capacity when they become less capable of proliferating, for example, because they undergo apoptosis or other forms of death, are unable to carry out cell cycles and/or divide, or differentiate. Non-limiting illustrative cytolytic agents include, but are not limited to, radioisotopes, photosensitizers (PS), cytotoxins, and chemotherapeutic agents.

在一些實施例中，本文所提供之融合蛋白之FAP結合部分可與一或多種放射性同位素（本文亦稱為放射性核種）連接、融合、或接合。例示性放射性核種包括：β粒子、α粒子、或俄歇電子發射體。適合的β發射體係例如釔-90、碘-131、鍶-89-氯化物、鎦-177、鈥-166、錸-186、錸-188、銅-67、鉕-149、金-199、及銠-105。適合的俄歇電子發射體例如係溴-77、銦-111、碘-123、及碘-125。適合的α發射體例如係釷-227、鉍-213、鐳-223、錒-225、及砈-211。In some embodiments, the FAP-binding portion of the fusion protein provided herein may be linked, fused with, or conjugated to one or more radioisotopes (also referred to herein as radionuclides). Exemplary radionuclides include beta particles, alpha particles, or Auger electron emitters. Suitable beta emitters include, for example, yttrium-90, iodine-131, strontium-89-chloride, indium-177, holmium-166, rhenium-186, rhenium-188, copper-67, argon-149, gold-199, and rhodium-105. Suitable Auger electron emitters include, for example, bromine-77, indium-111, iodine-123, and iodine-125. Suitable alpha emitters include thorium-227, bismuth-213, radium-223, azurium-225, and pyroxene-211.

例如，釷-227 (227Th)可與八齒3,2-羥基吡啶酮(3,2-HOPO)螯合劑有效地錯合，該八齒3,2-羥基吡啶酮(3,2-HOPO)螯合劑接合至根據本揭露之融合蛋白的FAP結合部分，產生高度穩定的標靶釷-227接合物(TTC)。標靶釷接合物(TTC)包含三個主要建構組元。錒-227的β粒子衰變後，第一建構組元、發射α-粒子的放射性核種227Th以離子交換層析法純化。第二建構組元係螯合劑，諸如鐵載蛋白衍生的螯合劑，其含有在利用用於生物接合的羧酸連接子官能化的對稱多胺支架上帶有四個3-羥基-N-甲基-2-吡啶酮部份的HOPO基團。與靶向部份的接合可透過與離胺酸殘基的ε-胺基形成醯胺鍵來實現。此等八齒3,2-HOPO螯合劑可用227Th有效地標記，在環境條件下具有高產率、純度、及穩定性。與通常需要加熱的四氮雜環十二烷-1,4,7,10-四乙酸(DOTA)螯合劑相比，HOPO螯合劑因為在環境溫度下有效的放射性標記及形成的錯合物的高穩定性而具有優越性。第三建構組元係靶向部份，亦即本文所提供之融合蛋白之FAP結合部分。For example, thorium-227 (227Th) can be effectively chelated with an octadentate 3,2-hydroxypyridinone (3,2-HOPO) chelator, which binds to the FAP-binding portion of the fusion protein according to the present disclosure, to produce a highly stable targeted thorium-227 conjugate (TTC). The targeted thorium conjugate (TTC) comprises three main building blocks. After the β-particle decay of thorium-227, the first building block, the radioactive nucleus 227Th emitting α-particles, is purified by ion exchange chromatography. The second building block is a chelating agent, such as a ferroprotein-derived chelating agent, containing four HOPO groups on a symmetrical polyamine scaffold functionalized with carboxylic acid linkers for bioconjugation. Binding to the target site is achieved by forming an amide bond with the ε-amino group of the lysine residue. These octanoic 3,2-HOPO chelating agents can be efficiently labeled with 227Th, exhibiting high yield, purity, and stability under ambient conditions. Compared to tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelating agents, which typically require heating, HOPO chelating agents are superior due to their efficient radiolabeling at ambient temperatures and the high stability of the resulting complexes. The third building block is the targeting portion, namely the FAP-binding portion of the fusion protein presented in this paper.

在一些實施例中，本文所提供之融合蛋白之FAP結合部分可與一或多種光敏劑(PS)連接、融合、或接合。光動力療法(photodynamic therapy, PDT)係一種非侵入性治療，其涉及PS在實體瘤中的積累，隨後局部遞送正確波長的光以引起PS的活化，這在氧存在下導致活性含氧物(reactive oxygen species, ROS)的原位產生，對細胞組分造成損傷，且最終導致壞死或細胞凋亡。PDT係腫瘤學中有前景的工具，但經常受到光敏劑靶向不足引起的副作用的限制。因此，本揭露涵蓋PS與腫瘤特異性結合劑（例如抗體）的接合。本文亦提供抗原結合Ab片段（例如Fab或scFv片段）的用途，因為在一些實施例中，抗原結合片段保留與全尺寸抗體相同的結合特異性，但因為其較小的尺寸而更有效地穿透腫瘤塊，且因為缺乏Fc域而更有效地從循環中清除。在一些實施例中，提供了用於光動力療法及光診斷領域的卟啉，且係生物醫學科學之此等領域中最突出的光敏劑類別中的一者(Sandland J, Boyle RW. Bioconjug Chem. 30(4):975-993 (2019))。在一些實施例中，光敏劑係四吡咯大環化合物。在一些實施例中，四吡咯大環化合物係卟啉、二氫卟酚、菌綠素、或酞菁。In some embodiments, the FAP-binding portion of the fusion protein provided herein may be linked, fused with, or conjugated to one or more photosensitizers (PS). Photodynamic therapy (PDT) is a non-invasive treatment involving the accumulation of PS in solid tumors, followed by local delivery of light of the correct wavelength to induce PS activation. This activation leads to the in situ production of reactive oxygen species (ROS) in the presence of oxygen, causing damage to cellular components and ultimately resulting in necrosis or apoptosis. PDT is a promising tool in oncology, but it is often limited by side effects due to insufficient photosensitizer targeting. Therefore, this disclosure covers the conjugation of PS to tumor-specific binders (e.g., antibodies). This article also provides the use of antigen-binding Ab fragments (such as Fab or scFv fragments) because, in some embodiments, antigen-binding fragments retain the same binding specificity as full-size antibodies, but penetrate tumor masses more effectively due to their smaller size and are cleared from circulation more effectively due to the lack of an Fc domain. In some embodiments, porphyrins are provided for use in photodynamic therapy and photodiagnosis, and are among the most prominent classes of photosensitizers in these fields of biomedical science (Sandland J, Boyle RW. Bioconjug Chem. 30(4):975-993 (2019)). In some embodiments, the photosensitizer is a tetrapyrrole macrocyclic compound. In some embodiments, the tetrapyrrole macrocyclic compound is porphyrin, dihydroporphyrin, chlorophyll, or phthalocyanine.

在一些實施例中，本文所提供之融合蛋白之FAP結合部分可與一或多種細胞毒性劑連接、融合、或接合，其在一些實施例中可來自抗體藥物接合物(ADC)，在本文中稱為抗FAP ADC。所屬技術領域中具有通常知識者可根據預期應用選擇適合的細胞毒素。在一些實施例中，細胞裂解劑係抗代謝劑、烷化劑、抗生素、生長因子、細胞介素、抗血管生成劑、抗有絲分裂劑、蒽環、毒素、或細胞凋亡劑中的至少一者。In some embodiments, the FAP-binding portion of the fusion protein provided herein may be linked, fused with, or conjugated to one or more cytotoxic agents, which in some embodiments may be derived from antibody-drug conjugates (ADCs), referred to herein as anti-FAP ADCs. Those skilled in the art can select a suitable cytotoxic agent according to the intended application. In some embodiments, the cytolytic agent is at least one of an antimetabolic agent, alkylating agent, antibiotic, growth factor, intercytokine, antiangiogenic agent, antimitotic agent, anthracycline, toxin, or apoptosis-inducing agent.

在一些實施例中，細胞毒性劑係奧瑞他汀、類美登素、驅動蛋白-紡錘體蛋白(kinesin-spindle protein, KSP)抑制劑、菸鹼醯胺磷酸核糖基轉移酶(nicotinamide phosphoribosyltransferase, NAMPT)抑制劑、或吡咯并苯二氮平衍生物。包含類美登素之接合物的產生可如Chari, Ravi V J, et al. Cancer research 52.1 (1992): 127-131、或EP2424569 B1中所述發生，兩者全部併入本文中。包含驅動蛋白-紡錘體蛋白(KSP)抑制劑之接合物的產生可如WO2019243159 A1中所述發生，該文獻全部併入本文中。包含菸鹼醯胺磷酸核糖基轉移酶(NAMPT)抑制劑之接合物的產生可如WO2019149637 A1中所述發生，該文獻全部併入本文中。包含吡咯并苯二氮平之接合物的產生可如EP3355935 A1中所述獲得，該文獻全部併入本文中。 In some embodiments, the cytotoxic agent is olistatin, maytansine-like substances, kinesin-spindle protein (KSP) inhibitors, nicotinamide phosphoribosyltransferase (NAMPT) inhibitors, or pyrrolobenzodiazepine derivatives. The formation of conjugates containing maytansine-like substances can occur as described in Chari, Ravi VJ, et al . Cancer research 52.1 (1992): 127-131, or EP2424569 B1, both of which are incorporated herein by reference. The formation of conjugates containing kinesin-spindle protein (KSP) inhibitors can occur as described in WO2019243159 A1, the entirety of which is incorporated herein by reference. The generation of conjugates containing niacinamide phosphoribosyltransferase (NAMPT) inhibitors can occur as described in WO2019149637 A1, the entire of which is incorporated herein by reference. The generation of conjugates containing pyrrolobenzodiazepine can be obtained as described in EP3355935 A1, the entire of which is incorporated herein by reference.

抗FAP ADC的細胞毒性劑及/或細胞生長抑制劑可係已知抑制細胞生長及/或複製、及/或殺滅細胞的任何試劑。許多具有細胞毒性及/或細胞生長抑制性質的試劑在文獻中係已知的。細胞毒性劑及/或細胞生長抑制劑類別的非限制性實例包括，舉實例而言但不限於，細胞週期調節劑、細胞凋亡調節劑、激酶抑制劑、蛋白質合成抑制劑、烷化劑、DNA交聯劑、嵌入劑、粒線體抑制劑、核輸出抑制劑、拓樸異構酶I抑制劑、拓樸異構酶II抑制劑、RNA/DNA抗代謝劑、及抗有絲分裂劑。Cytotoxic agents and/or cell growth inhibitors of anti-FAP ADCs can be any reagent known to inhibit cell growth and/or replication, and/or kill cells. Many reagents with cytotoxic and/or cell growth inhibitory properties are known in the literature. Non-limiting examples of the class of cytotoxic agents and/or cell growth inhibitors include, by way of example but not limited to, cell cycle regulators, apoptosis regulators, kinase inhibitors, protein synthesis inhibitors, alkylating agents, DNA crosslinking agents, intercalating agents, mitochondrial inhibitors, nuclear export inhibitors, topoisomerase I inhibitors, topoisomerase II inhibitors, RNA/DNA anti-metabolites, and antimitotic agents.

將（多種）細胞毒性劑及/或（多種）細胞生長抑制劑與抗FAP ADC的抗原結合部份連接的連接子本質上可係長的、短的、可撓性的、剛性的、親水性的、或疏水性的，或者可包含具有不同特性的區段，諸如可撓性區段、剛性區段等。連接子對於細胞外環境可係化學穩定的，例如在血流中化學穩定的，或者可包括不穩定的鍵聯且在細胞外環境中釋放細胞毒性劑及/或細胞生長抑制劑。在一些實施例中，連接子包括經設計以在抗FAP ADC內化到細胞內時釋放細胞毒性劑及/或細胞生長抑制劑的鍵聯。在一些具體實施例中，連接子包括經設計以在細胞內特異性或非特異性裂解及/或犧牲或以其他方式分解的鍵聯。可用於將藥物連接至抗原結合部份（諸如ADC背景下的抗體）的多種連接子係所屬技術領域中已知的。任何此等連接子以及其他連接子可用以將細胞毒性劑及/或細胞生長抑制劑與本文所述之抗FAP ADC的抗原結合部份連接。The linker that connects (multiple) cytotoxic agents and/or (multiple) cell growth inhibitors to the antigen-binding portion of the anti-FAP ADC can be inherently long, short, flexible, rigid, hydrophilic, or hydrophobic, or may contain segments with different properties, such as flexible segments, rigid segments, etc. The linker can be chemically stable to the extracellular environment, such as chemically stable in blood flow, or may include unstable links that release the cytotoxic agents and/or cell growth inhibitors in the extracellular environment. In some embodiments, the linker includes a linker designed to release a cytotoxic agent and/or a cell growth inhibitor upon internalization of the anti-FAP ADC into the cell. In some specific embodiments, the linker includes a linker designed to specifically or nonspecifically cleave and/or sacrifice or otherwise degrade within the cell. A variety of linkers that can be used to link a drug to an antigen-binding site (such as an antibody in the context of an ADC) are known in the art. Any such linker, and others, can be used to link a cytotoxic agent and/or a cell growth inhibitor to the antigen-binding site of the anti-FAP ADC described herein.

與抗FAP ADC的抗原結合部份連接的細胞毒性劑及/或細胞生長抑制劑的數目（藥物與抗體的比率(drug-to-antibody ratio)：DAR）可變化，且將僅受抗原結合部份上可用附著位點的數目及連接到單一連接子之試劑的數目的限制。一般而言，連接子將單一細胞毒性劑及/或細胞生長抑制劑連接至抗FAP ADC的抗原結合部份。在抗FAP ADC的實施例中，其包括多於一種細胞毒性劑及/或細胞生長抑制劑，各試劑可相同或不同。只要抗趨化因子受體或抗FAP ADC在使用及/或儲存條件下不展現出不可接受的聚集水平，則考慮具有二十或甚至更高的DAR的抗FAP ADC。在一些實施例中，本文所述之抗FAP ADC可具有在約1-10、1-8、1-6、或1-4範圍內的DAR。在某些具體實施例中，抗FAP ADC可具有2、3、或4的DAR。在一些實施例中，抗FAP ADC係根據結構式(1)的化合物： [D-L-XY] n-Ab 式1 或其鹽，其中各「D」獨立於其他代表細胞毒性劑及/或細胞生長抑制劑；各「L」獨立於其他代表連接子；「Ab」代表抗FAP受體結合部份，例如本文所提供之抗FAP抗體；各「XY」代表形成在連接子上的官能基Rx與抗趨化因子受體結合部份上的「互補」官能基Ry之間的鍵聯；且n代表抗趨化因子受體ADC的DAR。 The number of cytotoxic agents and/or cell growth inhibitors (drug-to-antibody ratio: DAR) linked to the antigen-binding site of the anti-FAP ADC can vary and will be limited only by the number of available attachment sites on the antigen-binding site and the number of reagents linked to a single linker. Generally, the linker links a single cytotoxic agent and/or cell growth inhibitor to the antigen-binding site of the anti-FAP ADC. In embodiments of the anti-FAP ADC, it includes more than one cytotoxic agent and/or cell growth inhibitor, which may be the same or different. Anti-FAP ADCs with a DAR of twenty or higher are considered, provided that the anti-chemotype receptor or anti-FAP ADC does not exhibit unacceptable aggregation levels under use and/or storage conditions. In some embodiments, the anti-FAP ADCs described herein may have a DAR in the range of about 1-10, 1-8, 1-6, or 1-4. In some specific embodiments, the anti-FAP ADC may have a DAR of 2, 3, or 4. In some embodiments, the anti-FAP ADC is a compound of structural formula (1): [DL-XY] n -Ab Formula 1 or a salt thereof, wherein each "D" is independent of the others representing a cytotoxic agent and/or a cell growth inhibitor; each "L" is independent of the others representing a linker; "Ab" represents the anti-FAP receptor binding site, such as the anti-FAP antibody provided herein; each "XY" represents the link between the functional group Rx formed on the linker and the "complementary" functional group Ry on the anti-chemotype receptor binding site; and n represents the DAR of the anti-chemotype receptor ADC.

在具體的例示性實施例中，抗FAP ADC係根據結構式(1)的化合物，其中各「D」係相同的且係細胞滲透性奧瑞他汀（例如尾海兔素-10或MMAE）或細胞滲透性小溝結合DNA交聯劑；各「L」係相同的且係可由溶酶體酶切割的連接子；各「XY」係形成在順丁烯二醯亞胺與氫硫基之間的鍵聯；「Ab」係包含對應於根據本揭露的抗趨化因子受體或FAP抗體之六個CDR的六個CDR的抗體或其片段；且n係2、3、或4。在具體實施例中，「Ab」係包含人源CDR的完全人類抗體。In a specific exemplary embodiment, the anti-FAP ADC is a compound according to structural formula (1), wherein each "D" is identical and is a cell-permeable olistatin (e.g., sulphurin-10 or MMAE) or a cell-permeable small groove DNA-binding crosslinker; each "L" is identical and is a linker cleavable by lysosomal enzymes; each "XY" is a link formed between a cis-diimidylamine and a hydrogen sulfide group; "Ab" is an antibody or fragment thereof containing six CDRs corresponding to six CDRs of an anti-chemofacial receptor or FAP antibody according to the present disclosure; and n is 2, 3, or 4. In a specific embodiment, "Ab" is a fully human antibody containing human CDRs.

細胞毒性劑及細胞生長抑制劑係已知抑制細胞，且特別係腫瘤細胞或腫瘤內Treg細胞的生長及/或複製及/或殺滅細胞的試劑。此等化合物可用於與抗趨化因子受體抗體（諸如FAP抗體）的組合療法，或作為本文所述之抗趨化因子受體ADC的一部分：在一些實施例中，抗趨化因子受體或抗FAP ADC的藥物部份係選自下列的細胞生長抑制劑：放射性核種、烷化劑、DNA交聯劑、DNA嵌入劑（例如溝結合劑，諸如小溝結合劑）、細胞週期調節劑、細胞凋亡調節劑、激酶抑制劑、蛋白質合成抑制劑、粒線體抑制劑、核輸出抑制劑、拓樸異構酶I抑制劑、拓樸異構酶II抑制劑、RNA/DNA抗代謝劑、及抗有絲分裂劑。在一些實施例中，抗趨化因子受體或抗FAP ADC的藥物部份係選自下列的烷化劑：asaley（L-白胺酸、N-[N-乙醯基-4-[雙-(2-氯乙基)胺基]-DL-苯丙胺醯基]-、乙酯）；AZQ（1,4-環己二烯-1,4-二胺基甲酸、2,5-雙(1-氮丙啶基)-3,6-二側氧基-,二乙酯）；BCNU（N,N'-雙(2-氯乙基)-N-亞硝基脲）；白消安（1,4-丁二醇二甲磺酸酯）；(羧基鄰苯二甲酸)鉑；CBDCA（順-(1,1-環丁烷二羧酸基)二胺鉑(II))）；CCNU（N-(2-氯乙基)-N'-環己基-N-亞硝基脲）；CHIP（異丙鉑；NSC 256927）；氯芥苯丁酸；吡葡亞硝脲（2-[[[(2-氯乙基)亞硝基胺基]羰基]胺基]-2-脫氧-D-吡喃葡萄糖）；順式鉑（順鉑）；克羅米松；氰 啉基阿黴素；環地鬆；雙脫水半乳糖醇（5,6-二環氧半乳糖醇）；氟多潘（5-[(2-氯乙基)-(2-氟乙基)胺基]-6-甲基-尿嘧啶）；庚磺安(hepsulfam)；海蒽酮(hycanthone)；吲哚并苯二氮平二聚體DGN462；美法侖；甲基CCNU（1-(2-氯乙基)-3-(反4-甲基環己烷)-1-亞硝基脲）；絲裂黴素C (mitomycin C)；米托唑胺；氮芥（雙(2-氯乙基)甲胺鹽酸鹽）；PCNU（1-(2-氯乙基)-3-(2,6-二側氧基-3-哌啶基)-1-亞硝基脲）；哌𠯤烷基化劑（1-(2-氯乙基)-4-(3-氯丙基)-哌𠯤二鹽酸鹽）；哌𠯤二酮；哌泊溴曼(pipobroman)（N,N'-雙(3-溴丙醯基)哌𠯤）；卟啉黴素（N-甲基絲裂黴素C）；螺乙內醯脲芥末；替羅昔隆(teroxirone)（三縮水甘油基異氰脲酸酯）；四鉑；噻替派(thio-tepa)（N,N',N''-三-1,2-乙二基硫代磷醯胺）；三乙烯三聚氰胺；尿嘧啶氮芥（去甲基多潘）；Yoshi-864（雙(3-甲磺氧基丙基)胺鹽酸鹽）。 Cytotoxic agents and cell growth inhibitors are known agents that inhibit, and particularly inhibit, the growth and/or replication and/or killing of tumor cells or intratumoral Treg cells. These compounds can be used in combination therapy with anti-chemofacial receptor antibodies (such as FAP antibodies), or as part of the anti-chemofacial receptor ADCs described herein: in some embodiments, the anti-chemofacial receptor or anti-FAP... The drug component of an ADC is selected from the following cell growth inhibitors: radioactive nuclei, alkylating agents, DNA crosslinking agents, DNA intercalating agents (e.g., groove binders, such as small groove binders), cell cycle regulators, apoptosis regulators, kinase inhibitors, protein synthesis inhibitors, mitochondrial inhibitors, nuclear export inhibitors, topoisomerase I inhibitors, topoisomerase II inhibitors, RNA/DNA anti-metabolites, and anti-mitotic agents. In some embodiments, anti-chemokine receptors or anti-FAP are also included. The drug fraction of an ADC is selected from the following alkylating agents: asaley (L-leucine, N-[N-acetylated-4-[bis-(2-chloroethyl)amino]-DL-phenylpropylamino]-, ethyl ester); AZQ (1,4-cyclohexadiene-1,4-diaminocarboxylic acid, 2,5-bis(1-aziridinyl)-3,6-dioxy-, diethyl ester); BCN U (N,N'-bis(2-chloroethyl)-N-nitrosourea); Busulfan (1,4-butanediol dimethyl sulfonate); (carboxylated phthalic acid)platinum; CBDCA (cis-(1,1-cyclobutanedicarboxylate)diamineplatinum(II))); CCNU (N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea); CHIP (isopropylplatinum; NSC 256927); chloromustine phenylbutyric acid; pyrogallol nitrosourea (2-[[[(2-chloroethyl)nitrosoamino]carbonyl]amino]-2-deoxy-D-glucopyranose); cisplatin (cisplatin); cromatasone; cyanide Linyl amycin; Cyclodesone; Dihydrogalactitol (5,6-diepoxygalactitol); Fluorodiphenyl ether (5-[(2-chloroethyl)-(2-fluoroethyl)amino]-6-methyluracil); Hepsulfam; Hycanthone; Indobenzobenzodiazepine dimer DGN462; Melphalan; Methyl CCNU (1-(2-chloroethyl)-3-(trans-4-methylcyclohexane)-1-nitrosourea); Mitomycin C C); mitoxazolamide; nitrogen mustard (bis(2-chloroethyl)methylaminohydrochloride); PCNU (1-(2-chloroethyl)-3-(2,6-dioxy-3-piperidinyl)-1-nitrosourea); piperazine alkylating agent (1-(2-chloroethyl)-4-(3-chloropropyl)-piperazine dihydrochloride); piperazine dione; pipobroman (N,N'-bis(3-bromopropyl)piperazine); porphyrin Phytosine (N-methylmitochondria C); Spiroyl urea mustard; Teroxirone (triglycidyl isocyanurate); Tetraplatin; Thiotepa (N,N',N''-tri-1,2-ethylenedimethylthiophosphatidylamine); Triethylene melamine; Uracil nitrogen mustard (desmethyldopan); Yoshi-864 (bis(3-methanesulfonoxypropyl)amine hydrochloride).

在一些實施例中，抗FAP ADC的藥物部份係選自下列的DNA烷化樣劑：順鉑；卡鉑；奈達鉑；奧沙利鉑；薩特拉鉑(Satraplatin)；四硝酸三鉑(Triplatin tetranitrate)；丙卡巴肼(Procarbazine)；六甲蜜胺；達卡巴仁；米托唑胺(mitozolomide)；替莫唑胺(temozolomide)。In some embodiments, the drug portion of the anti-FAP ADC is selected from the following DNA alkylating agents: cisplatin; carboplatin; nedaplatin; oxaliplatin; satraplatin; triplatin tetranitrate; procarbazine; hexamethylmelamine; dacarbazine; mitozolomide; temozolomide.

在一些實施例中，抗趨化因子受體或抗FAP ADC的藥物部份係選自下列的烷化抗贅瘤劑：卡波醌(Carboquone)；卡莫司汀(Carmustine)；萘氮芥(Chlornaphazine)；吡葡亞硝脲(Chlorozotocin)；雙聯黴素(Duocarmycin)；伊沃醯胺(Evofosfamide)；福莫司汀(Fotemustine)；葡磷醯胺(Glufosfamide)；洛莫司汀(Lomustine)；甘露硫丹(Mannosulfan)；尼氮芥(Nimustine)；菲鉑(Phenanthriplatin)；哌泊溴烷(Pipobroman)；雷莫司汀(Ranimustine)；司莫司汀(Semustine)；鏈脲黴素(Streptozotocin)；噻替哌(ThioTEPA)；曲硫丹(Treosulfan)；三亞胺醌(Triaziquone)；三乙烯三聚氰胺(Triethylenemelamine)；四硝酸三鉑。In some embodiments, the drug fraction for anti-chemotransferase receptors or anti-FAP ADCs is selected from the following alkylating antitumor agents: Carboquone; Carmustine; Chlornaphazine; Chlorozotocin; Duocarmycin; Evofosfamide; Fotemustine; Glufosfamide; Lomustine; Mannosulfuron. lfan); Nimustine; Phenanthriplatin; Pipobroman; Ranimustine; Semustine; Streptozotocin; ThioTEPA; Treosulfan; Triaziquone; Triethylenemelamine; Triplatinium tetranitrate.

在一些實施例中，抗FAP ADC的藥物部份係選自下列的DNA複製及修復抑製劑：六甲蜜胺；博來黴素(Bleomycin)；達卡巴仁；放線菌素D (Dactinomycin)；二溴甘露醇(Mitobronitol)；絲裂黴素；平陽黴素(Pingyangmycin)；普卡黴素(Pingyangmycin)；丙卡巴肼(Procarbazine)；替莫唑胺；ABT-888（維利帕尼(veliparib)）；奧拉帕尼(olaparib)；KU-59436；AZD-2281；AG-014699；BSI-201；BGP-15；INO-1001；ONO-2231.In some embodiments, the drug portion of the anti-FAP ADC is selected from the following DNA replication and repair inhibitors: hexamethylmelamine; bleomycin; dacarbazin; actinomycin D; mitobronitol; mitomycin; pingyangmycin; pingyangmycin; procarbazine; temozolomide; ABT-888 (veliparib); olaparib; KU-59436; AZD-2281; AG-014699; BSI-201; BGP-15; INO-1001; ONO-2231.

在一些實施例中，抗FAP ADC的藥物部份係細胞週期調節劑，諸如太平洋紫杉醇；Nab-太平洋紫杉醇；多西紫杉醇；長春新鹼；長春鹼；ABT-348；AZD-1152；MLN-8054；VX-680；Aurora A特異性激酶抑制劑；Aurora B特異性激酶抑制劑及泛Aurora激酶抑制劑；AZD-5438；BMI-1040；BMS-032；BMS-387；CVT-2584；黃酮吡啶醇(flavopyridol)；GPC-286199；MCS-5A；PD0332991；PHA-690509；塞利西利布(seliciclib)（CYC-202，R-羅斯科維汀）；ZK-304709；AZD4877、ARRY-520：GSK923295A。In some embodiments, the anti-FAP ADC drug component is a cell cycle regulator, such as paclitaxel; Nab-paclitaxel; docetaxel; vincristine; vinca; ABT-348; AZD-1152; MLN-8054; VX-680; Aurora A-specific kinase inhibitor; Aurora B-specific kinase inhibitors and pan-Aurora kinase inhibitors; AZD-5438; BMI-1040; BMS-032; BMS-387; CVT-2584; flavopyridol; GPC-286199; MCS-5A; PD0332991; PHA-690509; seliciclib (CYC-202, R-Roskovin); ZK-304709; AZD4877, ARRY-520: GSK923295A.

在一些實施例中，抗FAP ADC的藥物部份係細胞凋亡調節劑，諸如AT-101（(-)棉子酚）；G3139或奧布利默森(oblimersen)（靶向Bcl-2的反義寡核苷酸）；IPI-194；IPI-565；N-(4-(4-((4'-氯(1,1'-聯苯)-2-基)甲基)哌𠯤-1-基苯甲醯基)-4-(((1R)-3-(二甲胺基)-1-((苯硫基)甲基)丙基)胺基)-3-硝基苯磺醯胺)；N-(4-(4-((2-(4-氯苯基)-5,5-二甲基-1-環己-1-烯-1-基)甲基)哌𠯤-1-基)苯甲醯基)-4-(((1R)-3-( 啉-4-基)-1-((苯硫基)甲基)丙基)胺基)-3-((三氟甲基)磺醯基)苯磺醯胺；GX-070（Obatoclax ®；1H-吲哚、2-(2-((3,5-二甲基-1H-吡咯-2-基)亞甲基)-3-甲氧基-2H-吡咯-5-基)-))；HGS1029；GDC-0145；GDC-0152；LCL-161；LBW-242；維奈托克；靶向TRAIL或死亡受體（例如DR4及DR5）的藥劑，諸如ETR2-ST01、GDC0145、HGS-1029、LBY-135、PRO-1762；靶向凋亡蛋白酶、凋亡蛋白酶調節劑、BCL-2家族成員、死亡域蛋白質、TNF家族成員、Toll家族成員、及/或NF-κ-B蛋白質的藥物。 In some implementations, anti-FAP The drug component of ADCs is an apoptosis regulator, such as AT-101 ((-)gossypol); G3139 or oblimersen (an antisense oligonucleotide targeting Bcl-2); IPI-194; IPI-565; N-(4-(4-((4'-chloro(1,1'-biphenyl)-2-yl)methyl)piperazine-1-ylbenzoyl)-4-(((1R)-3-(dimethylamino)-1-((phenylthio)methyl)propyl)amino)-3-nitrobenzenesulfonamide); N-(4-(4-((2-(4-chlorophenyl)-5,5-dimethyl-1-cyclohexyl-1-en-1-yl)methyl)piperazine-1-yl)benzoyl)-4-(((1R)-3-( (Lin-4-yl)-1-((phenylthio)methyl)propyl)amino)-3-((trifluoromethyl)sulfonylurea)benzenesulfonamide; GX-070 (Obatoclax® ) ; 1H-indole, 2-(2-((3,5-dimethyl-1H-pyrrolo-2-yl)methylene)-3-methoxy-2H-pyrrolo-5-yl)-)); HGS1029; GDC-0145; GDC-0152; LCL-161; LBW-242; venetoclax; agents targeting TRAIL or death receptors (e.g., DR4 and DR5), such as ETR2-ST01, GDC0145, HGS-1029, LBY-135, PRO-1762; drugs targeting apoptotic proteases, apoptotic protease regulators, BCL-2 family members, death domain proteins, TNF family members, Toll family members, and/or NF-κ-B proteins.

在一些實施例中，抗FAP ADC的藥物部份係血管生成抑制劑，諸如ABT-869；AEE-788；阿西替尼(AG-13736)；AZD-2171；CP-547,632；IM-862；哌加他米布(pegaptamib)；索拉非尼(sorafenib)；BAY43-9006；帕唑帕尼(pazopanib)(GW-786034)；瓦塔拉尼布(vatalanib) (PTK-787, ZK-222584)；舒尼替尼(sunitinib)；SU-11248；VEGF trap；凡德他尼(vandetanib)；ABT-165；ZD-6474；DLL4抑制劑。In some implementations, the anti-FAP ADC drug component is angiogenesis inhibitors, such as ABT-869; AEE-788; axitinib (AG-13736); AZD-2171; CP-547,632; IM-862; pegaptamib; sorafenib; BAY43-9006; pazopanib (GW-786034); vatalanib (PTK-787, ZK-222584); sunitinib; SU-11248; VEGF trap; vandetanib; ABT-165; ZD-6474; and DLL4 inhibitors.

在一些實施例中，抗FAP ADC的藥物部份係蛋白酶體抑制劑，諸如硼替佐米(Bortezomib)；卡非佐米(Carfilzomib)；環氧黴素(Epoxomicin)；伊沙佐米(Ixazomib)；鹽孢醯胺A (Salinosporamide A)。In some embodiments, the drug component of the anti-FAP ADC is a proteasome inhibitor, such as bortezomib; carfilzomib; epoxomicin; ixazomib; and salinosporamide A.

在一些實施例中，抗趨化因子受體或抗FAP ADC的藥物部份係激酶抑制劑，諸如阿法替尼(Afatinib)；阿西替尼(Axitinib)；伯舒替尼(Bosutinib)；克唑替尼(Crizotinib)；達沙替尼(Dasatinib)；埃羅替尼(Erlotinib)；福他替尼(Fostamatinib)；吉非替尼(Gefitinib)；依魯替尼(Ibrutinib)；伊馬替尼(Imatinib)；拉帕替尼(Lapatinib)；樂伐替尼(Lenvatinib)；穆布替尼(Mubritinib)；尼洛替尼(Nilotinib)；帕唑帕尼(Pazopanib)；哌加他尼(Pegaptanib)；索拉非尼；舒尼替尼；SU6656；凡德他尼；維羅非尼(Vemurafenib)；CEP-701（來他替尼(lesaurtinib)）；XL019；INCB018424（魯索替尼(ruxolitinib)）；ARRY-142886（塞來替尼(selemetinib)）；ARRY-438162（畢尼替尼(binimetinib)）；PD-325901；PD-98059；AP-23573；CCI-779；依維莫司(everolimus)；RAD-001；雷帕黴素(rapamycin)；替西羅莫司(temsirolimus)；ATP競爭性TORC1/TORC2抑制劑，包括PI-103、PP242、PP30、Torin 1；LY294002；XL-147；CAL-120；ONC-21；AEZS-127；ETP-45658；PX-866；GDC-0941；BGT226；BEZ235；XL765。In some embodiments, the drug fraction for anti-chemokine receptors or anti-FAP ADCs is a kinase inhibitor, such as afatinib; axitinib; bosutinib; crizotinib; dasatinib; erlotinib; fostamatinib; gefitinib; ibrutinib; imatinib; lapatinib; lenvatinib; mubritinib; nilotinib; pazopanib; pegaptanib; sorafenib; sunitinib; SU665 6; Vandetanib; Vemurafenib; CEP-701 (lesaurtinib); XL019; INCB018424 (ruxolitinib); ARRY-142886 (selemetinib); ARRY-438162 (binimetinib); PD-325901; PD-98059; AP-23573; CCI-779; everolimus; RAD-001; rapamycin; temsirolimus; ATP-competitive TORC1/TORC2 inhibitors, including PI-103, PP242, PP30, Torin 1; LY294002;

在一些實施例中，抗FAP ADC的藥物部份係蛋白質合成抑制劑，諸如鏈黴素；二氫鏈黴素(Dihydrostreptomycin)；新黴素(Neomycin)；新黴素B (Framycetin)；巴龍黴素(Paromomycin)；核糖黴素(Ribostamycin)；卡那黴素(Kanamycin)；阿米卡星(Amikacin)；阿貝卡星(Arbekacin)；貝卡那黴素(Bekanamycin)；地貝卡星(Dibekacin)；妥布黴素(Tobramycin)；壯觀黴素(Spectinomycin)；潮黴素B (Hygromycin B)；巴龍黴素(Paromomycin)；見大黴素(Gentamicin)；奈替米星(Netilmicin)；西索米星(Sisomicin)；異帕米星(Isepamicin)；維爾黴素(Verdamicin)；阿斯特黴素(Astromicin)；四環素(Tetracycline)；多西環素(Doxycycline)；金黴素(Chlortetracycline)；氯黴素(Clomocycline)；地美環素(Demeclocycline)；萊甲環素(Lymecycline)；甲氯環素(Meclocycline)；美他環素(Metacycline)；米諾環素(Minocycline)；土黴素(Oxytetracycline)；青黴素(Penimepicycline)；羅利四環素(Rolitetracycline)；四環素(Tetracycline)；甘胺醯環素類(Glycylcyclines)；替加環素(Tigecycline)；㗁唑啶酮(Oxazolidinone)；依培唑胺(Eperezolid)；利奈唑胺(Linezolid)；泊西唑胺(Posizolid)；拉德唑胺(Radezolid)；蘭貝唑胺(Ranbezolid)；蘇替唑胺(Sutezolid)；泰地唑胺(Tedizolid)；肽基轉移酶抑制劑；氯黴素(Chloramphenicol)；阿齊達芬尼考(Azidamfenicol)；甲磺氯黴素(Thiamphenicol)；氟苯尼考(Florfenicol)；截短側耳素類(Pleuromutilins)；瑞他帕林(Retapamulin)；泰妙菌素(Tiamulin)；伐尼莫林(Valnemulin)；阿奇黴素(Azithromycin)；克拉黴素(Clarithromycin)；地紅黴素(Dirithromycin)；紅黴素(Erythromycin)；氟黴素(Flurithromycin)；交沙黴素(Josamycin)；麥迪黴素(Midecamycin)；米奧卡黴素(Miocamycin)；竹桃黴素(Oleandomycin)；羅卡黴素(Rokitamycin)；羅紅黴素(Roxithromycin)；螺旋黴素(Spiramycin)；醋竹桃黴素(Troleandomycin)；泰樂菌素(Tylosin)；酮內酯類(Ketolides)；泰利黴素(Telithromycin)；賽紅黴素(Cethromycin)；索紅黴素(Solithromycin)；克林黴素(Clindamycin)；林可黴素(Lincomycin)；吡利黴素(Pirlimycin)；鏈陽菌素(Streptogramins)；普那黴素(Pristinamycin)；奎奴普汀(Quinupristin)/達福普汀(dalfopristin)；維吉尼亞黴素(Virginiamycin)。In some embodiments, the drug component of the anti-FAP ADC is a protein synthesis inhibitor, such as streptomycin; dihydrostreptomycin; neomycin; framycetin; paromomycin; ribostamycin; kanamycin; amikacin; arbekacin; bekanamycin; dibekacin; tobramycin; spectinomycin; and hygromycin B. B); Paromomycin; Gentamicin; Netilmicin; Sisomicin; Isepamicin; Verdamicin; Astromicin; Tetracycline; Doxycycline; Chlortetracycline; Clomocycline; Demeclocycline; Lymecycline; Meclocycline; Metacycline; Minocycline; Oxycycline Tetracycline; Penimepicycline; Rolitetracycline; Tetracycline; Glycylcyclines; Tigecycline; Oxazolidinone; Eperezolid; Linezolid; Posizolid; Radezolid; Ranbezolid; Sutezolid; Tedizolid; Peptidyl transferase inhibitors; Chloramphenicol; Azidamfenico l); Thiamphenicol; Florfenicol; Pleuromutilins; Retapamulin; Tiamulin; Valnemulin; Azithromycin; Clarithromycin; Dirithromycin; Erythromycin; Flurhithromycin; Josamycin; Midecamycin; Miocamycin; Oleandomycin; Rokitamycin Roxithromycin; Spiramycin; Troleandomycin; Tylosin; Ketolides; Telithromycin; Cethromycin; Solithromycin; Clindamycin; Lincomycin; Pirlimycin; Streptogramins; Pristinamycin; Quinupristin/dalfopristin; Virginiamycin.

在一些實施例中，抗FAP ADC的藥物部份係組蛋白去乙醯化酶抑制劑，諸如伏立諾他(Vorinostat)；羅米地辛(Romidepsin)；西達本胺(Chidamide)；帕比司他(Panobinostat)；丙戊酸；貝林司他(Belinostat)；莫西替他(Mocetinostat)；阿貝司他(Abexinostat)；恩替司他(Entinostat)；SB939（普拉西諾他(pracinostat)）；瑞米諾他(Resminostat)；吉韋司他(Givinostat)；奎西諾他(Quisinostat)；硫脲基丁腈(Kevetrin ™)；CUDC-10；CHR-2845（替非司他(tefinostat)）；CHR-3996；4SC-202；CG200745；ACY-1215（羅西林司他(rocilinostat)）；ME-344；蘿蔔硫素(sulforaphane)。 In some embodiments, the drug component of the anti-FAP ADC is a histone deacetylase inhibitor, such as vorinostat; romidepsin; chidamide; panobinostat; valproic acid; belinostat; mocetinostat; abexinostat; entinostat; SB939 (pracinostat); resminostat; gemcitabine; quinsinostat; thiourea-butyronitrile (Kevetrin ™) . ); CUDC-10; CHR-2845 (teefinostat); CHR-3996; 4SC-202; CG200745; ACY-1215 (rocilinostat); ME-344; sulforaphane.

在一些實施例中，抗FAP ADC的藥物部份係拓樸異構酶I抑制劑，諸如喜樹鹼(camptothecin)；各種喜樹鹼衍生物及類似物（例如NSC 100880、NSC 603071、NSC 107124、NSC 643833、NSC 629971、NSC 295500、NSC 249910、NSC 606985、NSC 74028、NSC 176323、NSC 295501、NSC 606172、NSC 606173、NSC 610458、NSC 618939、NSC 610457、NSC 610459、NSC 606499、NSC 610456、NSC 364830、及NSC 606497）； 啉異氧紅黴素(morpholinisoxorubicin)；SN-38。 In some embodiments, the drug component of the anti-FAP ADC is a topoisomerase I inhibitor, such as camptothecin; various camptothecin derivatives and analogues (e.g., NSC 100880, NSC 603071, NSC 107124, NSC 643833, NSC 629971, NSC 295500, NSC 249910, NSC 606985, NSC 74028, NSC 176323, NSC 295501, NSC 606172, NSC 606173, NSC 610458, NSC 618939, NSC 610457, NSC 610459, NSC...). 606499, NSC 610456, NSC 364830, and NSC 606497); Morpholinisoxorubicin; SN-38.

在一些實施例中，抗FAP ADC的藥物部份係拓樸異構酶II抑制劑，諸如阿黴素(doxorubicin)；胺萘非特(amonafide)（苯并異喹啉二酮）；m-AMSA（4'-(9-吖啶基胺基)-3'-甲氧基甲磺醯苯胺）；蒽吡唑衍生物(NSC 355644)；依託泊苷(VP-16)；吡唑并吖啶（吡唑并[3,4,5-kl]吖啶-2(6H)-丙胺、9-甲氧基-N,N-二甲基-5-硝基-、單甲磺酸酯）；比生群鹽酸鹽；道諾黴素(daunorubicin)；去氧阿黴素(deoxydoxorubicin)；米托蒽醌(mitoxantrone)；甲諾加利(menogaril)；N,N-二芐道諾黴素；氧蒽唑；魯比達酮(rubidazone)；替尼泊苷(teniposide)。In some embodiments, the drug component of the anti-FAP ADC is a topoisomerase II inhibitor, such as doxorubicin; amonafide (benzo[a]isoquinolinedione); m-AMSA (4'-(9-acridylamino)-3'-methoxymethanesulfonylaniline); anthraquinone derivatives (NSC) 355644); Etoposide (VP-16); Pyrazoloacridine (pyrazolo[3,4,5-kl]acridine-2(6H)-propylamine, 9-methoxy-N,N-dimethyl-5-nitro-monosulfonate); Bismuth subhydrochloride; Daunorubicin; Deoxydoxorubicin; Mitoxantrone; Menogaril; N,N-dibenzyldoxantrone; Oxantrazole; Rubidazone; Teniposide.

在一些實施例中，抗FAP ADC的藥物部份係DNA嵌入劑，諸如蒽黴素(anthramycin)；奇卡黴素A (chicamycin A)；茅黴素(tomaymycin)；DC-81；西伯黴素(sibiromycin)；吡咯并苯二氮平衍生物；SGD-1882（(S)-2-(4-胺基苯基)-7-甲氧基-8-(3 S)-7-甲氧基-2-(4-甲氧基苯基)-5-側氧基-5,11a-二氫-1H-苯并[e]吡咯并[1,2-a][1,4]二氮呯-8-基)氧基)丙氧基)-1H-苯并[e]吡咯并[1,2-a][1,4]二氮呯-5(11aH)-酮）；SG2000（SJG-136；(11aS,11a'S)-8,8'-(丙烷-1,3-二基雙(氧基))雙(7-甲氧基-2-亞甲基-2,3-二氫-1H-苯并[e]吡咯并[1,2-a][1,4]二氮呯-5(11aH)-酮)）。In some embodiments, the drug component of the anti-FAP ADC is a DNA intercalating agent, such as anthramycin; chicamycin A; tomoymycin; DC-81; sibiromycin; pyrrolobenzodiazepine derivatives; SGD-1882 ((S)-2-(4-aminophenyl)-7-methoxy-8-(3-aminophenyl)-7-methoxy-8-(3-aminophenyl)-7-methoxy-8-methoxy ... S)-7-methoxy-2-(4-methoxyphenyl)-5-sideoxy-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazap-8-yl)oxy)propoxy)-1H-benzo[e]pyrrolo[1,2-a][1,4]diazap-5(11aH)-one); SG2000 (SJG-136; (11aS,11a'S)-8,8'-(propane-1,3-diylbis(oxy))bis(7-methoxy-2-methylene-2,3-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazap-5(11aH)-one).

在一些實施例中，抗FAP ADC的藥物部份係RNA/DNA抗代謝物，諸如L-丙胺酸；5-氮雜胞苷；5-氟尿嘧啶；阿西維星(acivicin)；胺基蝶呤衍生物N-[2-氯-5[[(2,4-二胺基-5-甲基-6-喹唑啉基)甲基]胺基]苯甲醯基]L-天冬胺酸(NSC 132483)；胺基蝶呤衍生物N-[4-[[(2,4-二胺基-5-乙基-6-喹唑啉基)甲基]胺基]苯甲醯基]L-天冬胺酸；胺基蝶呤衍生物N-[2-氯-4-[[(2,4-二胺基-6-蝶啶基)甲基]胺基]苯甲醯基]L-天冬胺酸一水合物；抗葉酸劑PT523（（Nα-(4-胺基-4-脫氧蝶醯基)-Nγ-半鄰苯二甲醯基-L-鳥胺酸））；貝克可溶性葉酸拮抗劑(NSC 139105)；二氯烯丙基羥萘醌（（2-(3, 3-二氯烯丙基)-3-羥基-1,4-萘醌）；布喹那(brequinar)；富多拉富(ftorafur)（（前驅藥；5-氟-1-(四氫-2-呋喃基)-尿嘧啶）；5,6-二氫-5-氮雜胞苷；胺甲喋呤；胺甲喋呤衍生物（N-[[4-[[(2,4-二胺基-6-蝶啶基)甲基]甲基胺基]-1-萘基]羰基]L-麩胺酸）；PALA（（N-(膦醯乙醯基)-L-天冬胺酸）；吡唑呋喃菌素(pyrazofurin)；曲美沙特(trimetrexate)。In some embodiments, the drug component of the anti-FAP ADC is an RNA/DNA antimetabolite, such as L-alanine; 5-azacytidine; 5-fluorouracil; acivicin; and the aminopterin derivative N-[2-chloro-5-[[(2,4-diamino-5-methyl-6-quinazolinyl)methyl]amino]benzoyl]L-aspartic acid (NSC). 132483); Aminopterin derivative N-[4-[[(2,4-diamino-5-ethyl-6-quinazolinyl)methyl]amino]benzoyl]L-aspartic acid; Aminopterin derivative N-[2-chloro-4-[[(2,4-diamino-6-pteridyl)methyl]amino]benzoyl]L-aspartic acid monohydrate; Antifolate PT523 ((Nα-(4-amino-4-deoxypteridyl)-Nγ-hemisphenadimethyl-L-guanine)); Bec soluble folic acid antagonist (NSC 139105); Dichloroallyl hydroxynaphthoquinone ((2-(3, 3-Dichloroallyl)-3-hydroxy-1,4-naphthoquinone); brequinar; ftorafur (a precursor drug; 5-fluoro-1-(tetrahydro-2-furanyl)-uracil); 5,6-dihydro-5-azacytidine; methotrexate; methotrexate derivatives (N-[[4-[[(2,4-diamino-6-pteridyl)methyl]methylamino]-1-naphthyl]carbonyl]L-glutamic acid); PALA (N-(phosphoacetyl)-L-aspartic acid); pyrazofurin; trimetrexate.

在一些實施例中，抗FAP ADC的藥物部份係DNA抗代謝物，諸如3-HP；2'-脫氧-5-氟尿苷；5-HP；α-TGDR（α-2'-脫氧-6-硫代鳥苷）；甘胺酸蚜腸菌素；ara C（胞嘧啶阿拉伯糖苷）；5-氮雜-2'-脫氧胞苷；β-TGDR（β-2'-脫氧-6-硫代鳥苷）；環胞苷；胍唑；羥基脲；肌苷糖二醛；麥剋菌素II；吡唑并咪唑；硫鳥嘌呤；硫嘌呤。In some embodiments, the drug component of the anti-FAP ADC is a DNA antimetabolite, such as 3-HP; 2'-deoxy-5-fluorouridine; 5-HP; α-TGDR (α-2'-deoxy-6-thioguanidine); glycine oxaliplatin; ara C (cytosine arabinoside); 5-aza-2'-deoxycytidine; β-TGDR (β-2'-deoxy-6-thioguanidine); cyclocytidine; guanidine; hydroxyurea; inosine dialdehyde; mitoxin II; pyrazoimidazole; thioguanine; thiopurine.

在一些實施例中，抗FAP ADC的藥物部份係粒線體抑制劑，諸如水鬼蕉鹼(pancratistatin)；苯潘他汀(phenpanstatin)；玫瑰紅-123；依地福辛(edelfosine)；d-α-生育醇琥珀酸酯；化合物11β；阿斯匹靈；玫瑰樹鹼(ellipticine)；小蘗鹼(berberine)；淺藍素(cerulenin)；GX015-070（Obatoclax ®；1H-吲哚、2-(2-((3,5-二甲基-1H-吡咯-2-基)亞甲基)-3-甲氧基-2H-吡咯-5-基)-）；南蛇藤醇(celastrol)（雷公藤紅素(tripterine)）；二甲雙胍；孔雀綠(Brilliant green)；ME-344。 In some embodiments, the drug component of the anti-FAP ADC is a mitochondrial inhibitor, such as pancratistatin; phenpanstatin; rose red-123; edelfosine; d-α-tocopherol succinate; compound 11β; aspirin; ellipticine; berberine; cerulenin; GX015-070 ( Obatoclax® ; 1H-indole, 2-(2-((3,5-dimethyl-1H-pyrrolo-2-yl)methylene)-3-methoxy-2H-pyrrolo-5-yl)-); celastrol (tripterine); metformin; brilliant green; ME-344.

在一些實施例中，抗FAP ADC的藥物部份係抗有絲分裂劑，諸如別秋水仙鹼(allocolchicine)；奧瑞他汀，諸如MMAE（單甲基奧瑞他汀E）及MMAF（單甲基奧瑞他汀F）；軟海綿素B (halichondrin B)；西馬多丁(cemadotin)；秋水仙鹼(colchicine)；秋水仙鹼衍生物（N-苯甲醯基-去乙醯基苯甲醯胺）；多拉司他汀-10；多拉司他汀-15；美登素；類美登素，諸如DM1（N2'-去乙醯基-N2'-(3-巰基-1-側氧基丙基)-美登素）；羅佐辛(rhozoxin)；太平洋紫杉醇；太平洋紫杉醇衍生物（（2'-N-[3-(二甲基胺基)丙基]戊二酸太平洋紫杉醇）；多西紫杉醇；硫代秋水仙鹼；三苯甲基半胱胺酸；硫酸長春鹼；硫酸長春新鹼。In some embodiments, the drug component of the anti-FAP ADC is an antimitotic agent, such as allocolchicine; olprestatins, such as MMAE (monomethylolprestatin E) and MMAF (monomethylolprestatin F); and halichondrin B. B); cemadotin; colchicine; colchicine derivatives (N-benzoyl-deacetylated benzoylamine); dolalastatin-10; dolalastatin-15; maytansine; maytansine-like compounds, such as DM1 (N2'-deacetylated-N2'-(3-phenyl-1-sideoxypropyl)-matansine); rhozoxin; paclitaxel; paclitaxel derivatives ((2'-N-[3-(dimethylamino)propyl]glutaric acid paclitaxel); docetaxel; thiocolchicine; triphenylmethylcysteine; vincristine sulfate; vincristine sulfate.

在一些實施例中，抗FAP ADC的藥物部份係核輸出抑制劑，諸如卡利他汀A (callystatin A)；去乳黴素(delactonmycin)；KPT-185（丙-2-基(Z)-3-[3-[3-甲氧基-5-(三氟甲基)苯基]-1,2,4-三唑-1-基]丙-2-烯酸酯）；卡祖黴素A (kazusamycin A)；瘦他汀(leptolstatin)；瘦呋喃A (leptofuranin A)；細黴素B (leptomycin B)；拉賈多內(ratjadone)；凡迪尼索(Verdinexor)（(Z)-3-[3-[3,5-雙(三氟甲基)苯基]-1,2,4-三唑-1-基]-N-吡啶-2-基丙-2-烯醯肼）。In some embodiments, the drug component of the anti-FAP ADC is a nuclear output inhibitor, such as callystatin A; delactonmycin; KPT-185 (propyl-2-yl(Z)-3-[3-[3-methoxy-5-(trifluoromethyl)phenyl]-1,2,4-triazol-1-yl]propyl-2-enoate); kazusamycin A; leptolstatin; leptofuranin A; leptomycin B. B); ratjadone; Verdinexor ((Z)-3-[3-[3,5-bis(trifluoromethyl)phenyl]-1,2,4-triazol-1-yl]-N-pyridin-2-ylpropyl-2-enylhydrazine).

在一些實施例中，抗FAP ADC的藥物部份係荷爾蒙治療劑，諸如阿那曲唑(anastrozole)；依西美坦(exemestane)；阿佐昔芬(arzoxifene)；比卡魯胺(bicalutamide)；西曲瑞克(cetrorelix)；地加瑞克(degarelix)；地洛瑞林(deslorelin)；曲洛司坦(trilostane)；地塞米松(dexamethasone)；氟他胺(flutamide)；雷洛昔芬(raloxifene)；法屈唑(fadrozole)；托瑞米芬(toremifene)；氟維司群(fulvestrant)；來曲唑(letrozole)；福美坦(formestane)；糖皮質素(glucocorticoid)；多克骨化醇(doxercalciferol)；碳酸司維拉姆(sevelamer carbonate)；拉索昔芬(lasofoxifene)；醋酸亮丙瑞林(leuprolide acetate)；甲地孕酮(megesterol)；米非司酮(mifepristone)；尼魯米特(nilutamide)；泰莫西芬檸檬酸鹽(tamoxifen citrate)；阿巴瑞克(abarelix)；潑尼松(prednisone)；非那雄胺(finasteride)；利洛斯坦(rilostane)；布舍瑞林(buserelin)；黃體釋素(luteinizing hormone releasing hormone, LHRH)；組胺瑞林(Histrelin)；曲洛斯坦(trilostane)或莫德拉斯坦(modrastane)；福斯瑞林(fosrelin)；戈舍瑞林(goserelin)。In some implementations, the drug component of the anti-FAP ADC is a hormone therapy, such as anastrozole; exemestane; azoxifene; bicalutamide; cetrorelix; degarelix; deslorelin; trilostane; dexamethasone; flutamide; raloxifene; fadrozole; toremifene; fulvestrant; letrozole; formestane; glucocorticoid; doxercalciferol; sevelamer Carbonate); Lasofoxifene; Leuprolide acetate; Megesterol; Mifepristone; Nilutamide; Tamoxifen citrate; Abarelix; Prednisone; Finasteride; Rilostane; Buserelin; Luteinizing hormone releasing hormone (LHRH); Histrelin; Trilostane or Modrastane; Fosrelin; Goserelin.

在一些實施例中，抗FAP ADC包含與藥物部份接合之靶向第二目標的額外結合部份。在一些實施例中，抗FAP ADC包含雙特異性抗體，該雙特異性抗體包含與藥物部份接合之FAP抗原結合部份及靶向第二目標的第二抗原結合部份。在一些實施例中，第二抗原結合部份係薩西土珠單抗(sacituzumab)，藥物部份係戈維特坎(govitecan)，且第二目標係TROP2。在一些實施例中，第二抗原結合部份係泰舒圖單抗(tisotumab)，藥物部份係維多汀(vedotin)，且第二目標係組織因子。在一些實施例中，第二抗原結合部份係因福土單抗(enfortumab)，藥物部份係維多汀(vedotin)，且第二目標係Nectin4。在一些實施例中，第二抗原結合部份係本妥昔單抗(brentuximab)，藥物部份係維多汀(vedotin)，且第二目標係CD30。在一些實施例中，第二抗原結合部份係曲妥珠單抗(trastuzumab)，藥物部份係德魯替康(deruxtecan)，且第二目標係HER2。在一些實施例中，第二抗原結合部份係曲妥珠單抗(trastuzumab)，藥物部份係恩他新(emtansine)，且第二目標係HER2。在一些實施例中，第二抗原結合部份係保納珠單抗(polatuzumab)，藥物部份係維多汀(vedotin)，且第二目標係CD79。在一些實施例中，第二抗原結合部份係英妥珠單抗(inotuzumab)，藥物部份係奧唑米星(ozogamicin)，且第二目標係CD22。在一些實施例中，第二抗原結合部份係吉妥珠單抗(gemtuzumab)，藥物部份係奧唑米星(ozogamicin)，且第二目標係CD33。在一些實施例中，第二抗原結合部份係隆卡妥昔單抗(loncastuximab)，藥物部份係特西林(tesirine)，且第二目標係CD19。在一些實施例中，第二抗原結合部份係貝蘭單抗(belantamab)，藥物部份係莫福汀(mafodotin)，且第二目標係BCMA。在一些實施例中，第二抗原結合部份係米爾唯土西單抗(mirvetuximab)，藥物部份係索星(soravtansine)，且第二目標係FR⍺。在一些實施例中，第二抗原結合部份係莫昔土莫單抗(moxetumomab)，藥物部份係帕蘇多托克斯(pasudotox)，且第二目標係CD22。In some embodiments, the anti-FAP ADC includes an additional binding portion that binds to the drug portion and targets a second target. In some embodiments, the anti-FAP ADC includes a bispecific antibody comprising an FAP antigen-binding portion that binds to the drug portion and a second antigen-binding portion that targets a second target. In some embodiments, the second antigen-binding portion is sacituzumab, the drug portion is govitecan, and the second target is TROP2. In some embodiments, the second antigen-binding portion is tisotumab, the drug portion is vedotin, and the second target is tissue factor. In some embodiments, the second antigen-binding moiety is enfortumab, the drug portion is vedotin, and the secondary target is Nectin4. In some embodiments, the second antigen-binding moiety is brentuximab, the drug portion is vedotin, and the secondary target is CD30. In some embodiments, the second antigen-binding moiety is trastuzumab, the drug portion is deruxtecan, and the secondary target is HER2. In some embodiments, the second antigen-binding moiety is trastuzumab, the drug portion is emtansine, and the secondary target is HER2. In some embodiments, the secondary antigen-binding moiety is polatuzumab, the drug portion is vedotin, and the secondary target is CD79. In some embodiments, the secondary antigen-binding moiety is inotuzumab, the drug portion is ozogamicin, and the secondary target is CD22. In some embodiments, the secondary antigen-binding moiety is gemtuzumab, the drug portion is ozogamicin, and the secondary target is CD33. In some embodiments, the secondary antigen-binding moiety is loncastuximab, the drug portion is tesirine, and the secondary target is CD19. In some embodiments, the second antigen-binding moiety is belantamab, the drug portion is mafodotin, and the secondary target is BCMA. In some embodiments, the second antigen-binding moiety is mirvetuximab, the drug portion is soravtansine, and the secondary target is FR⍺. In some embodiments, the second antigen-binding moiety is moxetumomab, the drug portion is pasudotox, and the secondary target is CD22.

包括或可修飾為包括與抗體及/或結合片段附接的位點的此等試劑中之任一者可包括在抗FAP ADC中。Any of these reagents, including or modifiable to include sites for attachment to the antibody and/or binding fragment, may be included in an anti-FAP ADC.

其他轉譯後修飾包括標記或「標籤(tag)」序列，諸如肽，以促進純化。在一些實施例中，標記或標籤胺基酸序列係六組胺酸肽，諸如pQE載體中提供的標籤（參見例如QIAGEN, Inc.）等，其中的許多係市售的。例如，如Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-24, 1989中所描述的，六組胺酸提供了融合蛋白的方便純化。可用於純化的其他肽標籤包括但不限於血球凝集素（「HA」）標籤及「FLAG」標籤，該血球凝集素標籤對應於衍生自流感血球凝集素蛋白的表位(Wilson et al., Cell 37:767-78, 1984)。 Other post-translational modifications include marker or "tag" sequences, such as peptides, to facilitate purification. In some embodiments, the marker or tag amino acid sequence is a hexahistamine peptide, such as those provided in pQE vectors (see, for example, QIAGEN, Inc.), many of which are commercially available. For example, as described in Gentz et al ., Proc. Natl. Acad. Sci. USA 86:821-24, 1989, hexahistamine provides convenient purification of fusion proteins. Other peptide tags that can be used for purification include, but are not limited to, hemagglutinin ("HA") tags and "FLAG" tags, which correspond to epitopes derived from influenza hemagglutinin proteins (Wilson et al ., Cell 37:767-78, 1984).

用於將部份（包括多肽）（直接或間接）連接或接合至融合蛋白（例如抗FAP抗體、FAP抗原結合片段、或包含FAP抗原結合片段的雙特異性抗體）之FAP結合部分的方法係所屬技術領域中熟知的，其中之任一者可用以製備經修飾的FAP結合劑，包括本文所述之ADC及/或融合蛋白。 連接子 Methods for linking or conjugating portions (including peptides) (directly or indirectly) to the FAP-binding portion of a fusion protein (e.g., an anti-FAP antibody, a FAP antigen-binding fragment, or a bispecific antibody containing a FAP antigen-binding fragment) are well known in the art, and any of these methods can be used to prepare modified FAP binders, including the ADCs and/or fusion proteins described herein. Linker

本文提供一或多種連接子或其用途。在一些實施例中，本文所提供之融合蛋白包含一、2、3、4、5、或更多個連接子。在一些實施例中，融合蛋白中之各連接子可係不同的或可係相同的，或可係其組合。連接子可係肽連接子或合成連接子。在一些實施例中，連接子具有特定長度以影響融合蛋白之活性。連接子可經組態以驅動用於如本文所述之目標結合之二級結構的形成，諸如抗體組分的異二聚化。例如，連接子長度係短的以防止本文所述之融合蛋白的二或更多種組分之間的配對及/或以驅動本文所述之融合蛋白的二或更多種組分之間的配對。在另一實例中，連接子可係可撓性的。This document provides one or more linkers or their uses. In some embodiments, the fusion protein provided herein comprises one, two, three, four, five, or more linkers. In some embodiments, the linkers in the fusion protein may be different, identical, or combinations thereof. Linkers may be peptide linkers or synthetic linkers. In some embodiments, linkers have a specific length to influence the activity of the fusion protein. Linkers may be configured to drive the formation of secondary structures for target binding as described herein, such as heterodimerization of antibody components. For example, linkers may be short to prevent pairing between two or more components of the fusion protein described herein and/or to drive pairing between two or more components of the fusion protein described herein. In another embodiment, linkers may be flexible.

連接子的實例包括VH-CH1連接子ASTKGPSVFPLAPS (SEQ ID NO: 90)；VL-CL連接子RTVAAPSVFIFPPS (SEQ ID NO: 91)；CH2-CH3連接子ISKAKGQPREPQ (SEQ ID NO: 92)；IgM尾部連接子KSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 93)；GGGGSGGGGSGGGGSGGGGT (SEQ ID NO: 94)；G；及(GGGGS)n，n=l、2、3、4、5、6、7、8、9、或10，(SEQ ID NO: 95)。Examples of connectors include VH-CH1 connector ASTKGPSVFPLAPS (SEQ ID NO: 90); VL-CL connector RTVAAPSVFIFPPS (SEQ ID NO: 91); CH2-CH3 connector ISKAKGQPREPQ (SEQ ID NO: 92); IgM tail connector KSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 93); GGGGSGGGGSGGGGSGGGGT (SEQ ID NO: 94); G; and (GGGGS)n, n=1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (SEQ ID NO: 95).

本文描述了包含連接子A、B、及C之融合蛋白的形式。在一些實施例中，連接子A、連接子B、及連接子C獨立地係具有式(Gly4Ser) n的肽連接子，其中n係1、2、3、4、或5 (SEQ ID NO: 95)。在一些實施例中，n係2或3，亦即，連接子係(Gly4Ser) 2或(Gly4Ser) 3。在一些實施例中，連接子A、連接子B、及連接子C獨立地係胺基酸G。 This document describes a fusion protein comprising connectives A, B, and C. In some embodiments, connectives A, B, and C are independently peptide connectives having the formula (Gly4Ser) n , where n is 1, 2, 3, 4, or 5 (SEQ ID NO: 95). In some embodiments, n is 2 or 3, i.e., the connective is (Gly4Ser) 2 or (Gly4Ser) 3 . In some embodiments, connectives A, B, and C are independently amino acid G.

本揭露的融合蛋白可使用所屬技術領域中已知的方法產生。 修飾 The fusion protein disclosed herein can be produced using methods known in the art. Modification

在一些實施例中，將本文所提供之FAP結合劑用一或多個修飾工程改造。此類修飾可包括改變FAP結合劑之胺基酸序列導致一或多種胺基酸改變的修飾、及/或導致一或多種化學改變的轉譯後修飾。In some embodiments, the FAP binder provided herein is modified by one or more modification engineering processes. Such modifications may include modifications that alter the amino acid sequence of the FAP binder to result in one or more amino acid changes, and/or post-translational modifications that result in one or more chemical changes.

在一些實施例中，一或多個修飾包含一或多個胺基酸改變、一或多個化學改變、與一或多個第二試劑、一或多個連接子、或其任何組合的一或多個接合或融合。 胺基酸改變 In some embodiments, one or more modifications include one or more amino acid changes, one or more chemical changes, one or more interactions or fusions with one or more second reagents, one or more linkers, or any combination thereof. Amino acid changes

在一些實施例中，一或多個修飾包含一或多個胺基酸改變。在一些實施例中，將本文所提供之FAP結合劑用一、2、3、4、5、或更多個胺基酸改變工程改造。胺基酸改變包含一或多個胺基酸取代、缺失、及/或插入。在一些實施例中，一或多個胺基酸取代包含保守性取代或非保守性取代。對於作為FAP結合劑（諸如人類FAP結合劑）的多肽（例如抗體、片段、及/或結合多肽），保守性胺基酸取代包括其中胺基酸殘基經具有相似側鏈的胺基酸殘基置換的取代。所屬技術領域中已定義具有相似側鏈之胺基酸殘基的家族。此等家族包括具有鹼性側鏈之胺基酸（例如離胺酸、精胺酸、組胺酸）、酸性側鏈（例如天冬胺酸、麩胺酸）、未帶電極性側鏈（例如甘胺酸、天冬醯胺酸、麩醯胺酸、絲胺酸、蘇胺酸、酪胺酸、半胱胺酸、色胺酸）、非極性側鏈（例如丙胺酸、纈胺酸、白胺酸、異白胺酸、脯胺酸、苯丙胺酸、甲硫胺酸）、β-支鏈側鏈（例如蘇胺酸、纈胺酸、異白胺酸）、及芳族側鏈（例如酪胺酸、苯丙胺酸、色胺酸、組胺酸）。此類經保守修飾的變體係對同質多形變體、種間同系物、及等位基因的補充，且不排除同質多形變體、種間同系物、及等位基因。以下八組含有彼此保守取代的胺基酸：1)丙胺酸(A)、甘胺酸(G)；2)天冬胺酸(D)、麩胺酸(E)；3)天冬醯胺酸(N)、麩醯胺酸(Q)；4)精胺酸(R)、離胺酸(K)；5)異白胺酸(I)、白胺酸(L)、甲硫胺酸(M)、纈胺酸(V)；6)苯丙胺酸(F)、酪胺酸(Y)、色胺酸(W)；7)絲胺酸(S)、蘇胺酸(T)；及8)半胱胺酸(C)、甲硫胺酸(M)（參見，例如，Creighton, Proteins (1984)）。在一些實施例中，用語「保守序列修飾(conservative sequence modification)」用以指不顯著影響或改變含有胺基酸序列之抗體的結合特徵的胺基酸修飾。In some embodiments, one or more modifications involve one or more amino acid changes. In some embodiments, the FAP binder provided herein is engineered with one, two, three, four, five, or more amino acid changes. Amino acid changes include one or more amino acid substitutions, deletions, and/or insertions. In some embodiments, one or more amino acid substitutions include conservative substitutions or non-conservative substitutions. For peptides (e.g., antibodies, fragments, and/or binding peptides) that serve as FAP binders (such as human FAP binders), conservative amino acid substitutions include substitutions in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histamine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, aspartic acid, glutamic acid, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, succinic acid, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, succinic acid, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histamine). These conserved modified variants supplement homomorphic polymorphs, interspecific homologs, and alleles, and do not exclude homomorphic polymorphs, interspecific homologs, and alleles. The following eight groups contain amino acids that are conserved substitutes for each other: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) aspartic acid (N), glutamic acid (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valerine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine (C), methionine (M) (see, for example, Creighton, Proteins (1984)). In some embodiments, the term "conservative sequence modification" is used to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of an antibody containing an amino acid sequence.

因此，在一些實施例中，FAP結合劑中預測的非必需胺基酸殘基經來自同一側鏈家族的另一胺基酸殘基置換。識別不消除抗原結合的核苷酸及胺基酸保守性取代的方法係所屬技術領域中熟知的（參見例如Brummell et al., Biochem. 32:1180-1187 (1993)；Kobayashi et al. Protein Eng. 12(10):879-884 (1999)；及Burks et al. Proc. Natl. Acad. Sci. USA 94:412-417 (1997)）。在一些實施例中，本文所述之保守性胺基酸改變將FAP結合劑（例如抗體）（包括人類FAP結合劑）的胺基酸序列修飾50%、或55%、或60%、或65%、或70%、或75%、或80%、或85%、或90%、或95%、或98%、或99%。在一些實施例中，核苷酸及胺基酸取代係指 表 6或 表 7中描述之CDR的至多1、2、3、4、5、或6個胺基酸取代。因此，例如，各個此類CDR可含有至多5個保守性胺基酸取代，例如至多（不多於）4個保守性胺基酸取代，例如至多（不多於）3個保守性胺基酸取代，例如至多（不多於）2個保守性胺基酸取代、或不多於1個保守性胺基酸取代。 Therefore, in some embodiments, the predicted non-essential amino acid residue in the FAP binder is replaced by another amino acid residue from the same sidechain family. Methods for identifying nucleotides and conserved amino acid substitutions that do not eliminate antigen binding are well known in the relevant art (see, for example, Brummell et al ., Biochem. 32:1180-1187 (1993); Kobayashi et al . Protein Eng. 12(10):879-884 (1999); and Burks et al . Proc. Natl. Acad. Sci. USA 94:412-417 (1997)). In some embodiments, the conserved amino acid changes described herein modify the amino acid sequence of the FAP binder (e.g., antibody) (including human FAP binders) by 50%, or 55%, or 60%, or 65%, or 70%, or 75%, or 80%, or 85%, or 90%, or 95%, or 98%, or 99%. In some embodiments, nucleotide and amino acid substitutions refer to up to 1, 2, 3, 4, 5, or 6 amino acid substitutions in the CDRs described in Table 6 or Table 7. Thus, for example, each of these CDRs may contain up to 5 conserved amino acid substitutions, for example, up to (no more than) 4 conserved amino acid substitutions, for example, up to (no more than) 3 conserved amino acid substitutions, for example, up to (no more than) 2 conserved amino acid substitutions, or no more than 1 conserved amino acid substitution.

在一些實施例中，本文所提供之抗體係親和力成熟的，其中與不具有此類改變的親代抗體相比，此類抗體在一或多個CDR中包含一或多種胺基酸改變，此類改變導致抗體對抗原之親和力改善。可以使用適合的選擇及/或誘變方法使本文所提供之抗FAP抗體親和力成熟。在一些實施例中，親和力成熟抗體之親和力係製備成熟抗體之起始抗體（例如小鼠或兔人源化或人類抗體）之1.5倍或更多、2倍或更多、3倍或更多、4倍或更多、5倍或更多、10倍或更多、20倍或更多、或30倍或更多。In some embodiments, the antibodies provided herein are affinity-matured, wherein, compared to parental antibodies without such modifications, these antibodies contain one or more amino acid modifications in one or more CDRs, resulting in improved affinity of the antibody for the antigen. The affinity maturation of the anti-FAP antibodies provided herein can be achieved using suitable selection and/or mutagenesis methods. In some embodiments, the affinity of the affinity-matured antibody is 1.5 times or more, 2 times or more, 3 times or more, 4 times or more, 5 times or more, 10 times or more, 20 times or more, or 30 times or more than that of the starting antibody (e.g., humanized mouse or rabbit or human antibody) used to prepare the mature antibody.

在一些實施例中，本文所提供之FAP結合劑包含調節抗體體內半衰期的一或多個修飾。例如，解決Fc與FcRn的交互作用允許調節抗體的體內半衰期。在一些實施例中，藉由例如引入突變H435A來消除交互作用導致極短的半衰期，因為FcRn循環不再保護抗體免受溶酶體降解。在一些實施例中，本文所提供之FAP結合劑（例如抗體）包含修飾（包含H435A取代）或已以其他方式經工程改造以減少半衰期。In some embodiments, the FAP binders provided herein include one or more modifications that regulate the in vivo half-life of the antibody. For example, resolving the interaction between Fc and FcRn allows for regulation of the in vivo half-life of the antibody. In some embodiments, eliminating the interaction by, for example, introducing a mutation in H435A results in an extremely short half-life because the FcRn cycle no longer protects the antibody from lysosomal degradation. In some embodiments, the FAP binders (e.g., antibodies) provided herein include modifications (including H435A substitution) or have been otherwise engineered to reduce the half-life.

在一些實施例中，本文所述之FAP結合劑包含延長本文提供之抗FAP結合劑之半衰期的一或多個修飾。例如，已顯示包含「YTE」突變(M252Y/S254T/T256E)及/或諸如「LS」突變(M428L/N434S)的等效突變的抗體藉由更有效地從臨床前物種以及人類兩者中的內體循環而顯著延長半衰期(Dall'Acqua, William F., et al. The Journal of Immunology 169.9: 5171-5180 (2002); Zalevsky, Jonathan, et al“Enhanced antibody half-life improves in vivoactivity.” Nature biotechnology 28.2 (2010): 157-159.)。因此，在一些實施例中，本文所提供之FAP結合劑包含YTE突變(M252Y/S254T/T256E)及/或諸如LS (M428L/N434S)的等效突變，或者已經以其他方式經工程改造以改善半衰期。用於延長半衰期之適合的Fc工程改造方法可見於Haraya, Kenta, Tatsuhiko Tachibana, and Tomoyuki Igawa. Drug metabolism and pharmacokinetics 34.1: 25-41 (2019)及/或Lee, Chang-Han, et al. Nature communications 10.1: 1-11 (2019)，兩者皆以引用方式併入本文中。 In some embodiments, the FAP binders described herein include one or more modifications that extend the half-life of the anti-FAP binders provided herein. For example, antibodies containing equivalent mutations such as the "YTE" mutation (M252Y/S254T/T256E) and/or the "LS" mutation (M428L/N434S) have been shown to significantly extend half-life by more effectively circulating from endosomal sources in both preclinical and human species (Dall'Acqua, William F., et al . The Journal of Immunology 169.9: 5171-5180 (2002); Zalevsky, Jonathan, et al. "Enhanced antibody half-life improves in vivo activity." Nature biotechnology 28.2 (2010): 157-159.). Therefore, in some embodiments, the FAP binders described herein contain YTE mutations (M252Y/S254T/T256E) and/or equivalent mutations such as LS (M428L/N434S), or have been otherwise engineered to improve half-life. Suitable Fc engineering methods for prolonging half-life can be found in Haraya, Kenta, Tatsuhiko Tachibana, and Tomoyuki Igawa. Drug metabolism and pharmacokinetics 34.1: 25-41 (2019) and/or Lee, Chang-Han, et al . Nature communications 10.1: 1-11 (2019), both of which are incorporated herein by reference.

在一些實施例中，本文所提供之FAP結合劑包含促進Fc域的第一次單元及第二次單元締合的一或多個修飾。此類修飾包括肽主鏈的操作或Fc域次單元的轉譯後修飾，其減少或防止包含Fc域次單元的多肽與相同的多肽締合以形成同二聚體。包含Fc區的抗體可包含或可不包含促進Fc域的第一次單元及第二次單元締合的修飾。如本文中所使用的促進締合的修飾包括對所欲締合的兩個Fc域次單元（例如Fc域之第一次單元及第二次單元）之各者進行的單獨修飾，其中修飾係彼此互補以促進兩個Fc域次單元的締合。例如，促進締合的修飾可改變一或兩個Fc域次單元之結構或電荷，使其等在空間或靜電上有利地締合。因此，（異源）二聚化發生在包含第一Fc域次單元之多肽與包含第二Fc域次單元之多肽之間，其可能不相同，例如在與次單元之各者融合的其他組分（例如抗原結合部份）不相同的意義上。在一些實施例中，促進締合的修飾包含Fc域中的胺基酸改變，具體地係胺基酸取代。在特定的實施例中，促進締合的修飾包含Fc域之兩個次單元之各者中的單獨的胺基酸改變，具體地係胺基酸取代。 化學改變 In some embodiments, the FAP binders provided herein include one or more modifications that promote the bonding of the first and second units of the Fc domain. Such modifications include manipulations of the peptide backbone or post-translational modifications of the Fc domain subunits that reduce or prevent the bonding of the peptide containing the Fc domain subunits with the same peptide to form a homodimer. Antibodies containing the Fc region may or may not contain modifications that promote the bonding of the first and second units of the Fc domain. As used herein, bonding-promoting modifications include individual modifications to each of the two Fc domain subunits to be bonded (e.g., the first and second units of the Fc domain), wherein the modifications are complementary to each other to promote the bonding of the two Fc domain subunits. For example, a binding-promoting modification can alter the structure or charge of one or both Fc domain subunits, making them sterically or electrostatically compatible. Therefore, (hetero)dimerization occurs between a polypeptide containing a first Fc domain subunit and a polypeptide containing a second Fc domain subunit, which may be different, for example, in the sense that other components fused to each subunit (e.g., antigen-binding moieties) are different. In some embodiments, binding-promoting modifications involve amino acid changes in the Fc domain, specifically amino acid substitution. In certain embodiments, binding-promoting modifications involve individual amino acid changes in each of the two subunits of the Fc domain, specifically amino acid substitution. Chemical Change

在一些實施例中，本文所提供之FAP結合劑藉由一或多種化學改變來修飾，該一或多種化學改變包含糖基化（例如無岩藻糖基化(afucosylation)）、乙醯化、聚乙二醇化、磷酸化、硫酸化、醯胺化、已知保護/阻斷基團的衍生化、蛋白水解切割、與細胞配體或其他蛋白質的鍵聯。許多化學修飾中之任一者可由已知技術進行，包括但不限於特異性化學裂解、乙醯化、甲醯化、衣黴素的代謝合成等。在一些實施例中，藉由一或多種化學改變修飾的抗體在本文中被稱為衍生化抗體或衍生物。另外，衍生物可含有一或多種非天然胺基酸，例如，使用ambrx技術，參見例如Wolfson, Wendy. “Amber codon flashing ambrx augments proteins with unnatural amino acids.” Chemistry & biology 13.10 (2006): 1011-1012。In some embodiments, the FAP binders provided herein are modified by one or more chemical changes, including glycosylation (e.g., afucosylation), acetylation, polyethylene glycolation, phosphorylation, sulfation, amination, derivatization of known protecting/blocking groups, proteolytic cleavage, and binding to cellular ligands or other proteins. Any of these chemical modifications can be performed using known techniques, including but not limited to specific chemical cleavage, acetylation, methylation, and chlamydia metabolism. In some embodiments, antibodies modified by one or more chemical changes are referred to herein as derivatized antibodies or derivatives. In addition, derivatives may contain one or more non-natural amino acids, for example, using ambrx technology, see Wolfson, Wendy. “Amber codon flashing ambrx augments proteins with unnatural amino acids.” Chemistry & biology 13.10 (2006): 1011-1012.

在一些實施例中，本文所提供之FAP結合劑包含改變至少一個恆定區介導的生物效應功能的一或多個修飾。例如，在一些實施例中，相對於未經修飾的FAP結合劑，可以修飾FAP結合劑以降低或增強至少一種恆定區介導的生物效應功能，例如，減少或改善與Fc受體(FcγR)的結合。FcγR結合可能減少，例如藉由在FcγR交互作用所必需的特定區使抗體之免疫球蛋白恆定區區段突變（參見例如Canfield, Stephen M., and Sherie L. Morrison. The Journal of experimental medicine 173.6: 1483-1491 (1991)；及Lund, John, et al. The Journal of Immunology 147.8: 2657-2662 (1991)）。FcγR結合可例如藉由無岩藻糖基化增強。減少FcγR結合亦可能減少依賴FcγR交互作用的其他效應功能，諸如調理作用（例如CDC）、吞噬作用（例如ADCP）、及抗原依賴性細胞毒性（例如ADCC） In some embodiments, the FAP binders provided herein include one or more modifications that alter the function of at least one stationary region-mediated biological effect. For example, in some embodiments, the FAP binders may be modified, relative to the unmodified FAP binders, to reduce or enhance the function of at least one stationary region-mediated biological effect, such as reducing or improving binding to an Fc receptor (FcγR). FcγR binding may be reduced, for example, by mutation of immunoglobulin homeostasis regions of antibodies in specific regions essential for FcγR interactions (see, for example, Canfield, Stephen M., and Sherie L. Morrison. The Journal of Experimental Medicine 173.6: 1483-1491 (1991); and Lund, John, et al . The Journal of Immunology 147.8: 2657-2662 (1991)). FcγR binding can be enhanced, for example, by the absence of fucosylation. Reduced FcγR binding may also reduce other FcγR interaction-dependent effector functions, such as opsonization (e.g., CDC), phagocytosis (e.g., ADCP), and antigen-dependent cytotoxicity (e.g., ADCC).

因此，在一些實施例中，本文所提供之抗體及/或結合多肽經修飾，使得抗體的Fc區中的寡糖不具有任何岩藻糖單元或具有減少的岩藻糖單元（例如無岩藻糖基化的）。從附接至Fc上的雙觸角錯合物型寡糖中去除核心岩藻糖可大大增加ADCC效應功能，而不改變抗原結合或CDC效應功能。已知若干種減少或消除含Fc分子（例如抗體）的岩藻糖基化的方法。此等包括在某些哺乳動物細胞系中的重組表現，該等哺乳動物細胞系包括FUT8敲除細胞系、變體CHO系Lec13、大鼠融合瘤細胞系YB2/0、包含特異性針對FUT8基因之小干擾RNA的細胞系、及共表現α-1,4-N-乙醯葡糖胺基轉移酶III及高基氏α-甘露糖苷酶II的細胞系。替代地，含Fc的分子可在非哺乳動物細胞（諸如植物細胞、酵母菌、或原核細胞，例如大腸桿菌）中表現。鋅指核酸酶係產生無岩藻糖基化抗體的另一種已知方法。參見例如Haryadi et al., Bioengineered 4:2, 90-94 (2013)；Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986)；Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004)；Pereira et al. mAbs 10(5): 693-711 (2018)。 Therefore, in some embodiments, the antibodies and/or binding peptides provided herein are modified such that the oligosaccharides in the Fc region of the antibody lack any fucose units or have reduced fucose units (e.g., unfucosylated). Removing the core fucose from biantennary-wedge-type oligosaccharides attached to the Fc region can significantly increase ADCC efficacy without altering antigen-binding or CDC efficacy. Several methods for reducing or eliminating fucosylation in Fc-containing molecules (e.g., antibodies) are known. These include recombination expression in certain mammalian cell lines, including FUT8 knockout cell lines, the variant CHO line Lec13, the rat fusion tumor cell line YB2/0, cell lines containing small interfering RNA specifically targeting the FUT8 gene, and cell lines co-expressing α-1,4-N-acetylglucosamine transferase III and Gorgni's α-mannosidase II. Alternatively, Fc-containing molecules can be expressed in non-mammalian cells (such as plant cells, yeast, or prokaryotic cells, such as Escherichia coli). Another known method for producing fucosylated antibodies using zinc finger nuclease systems is... See, for example, Haryadi et al ., Bioengineered 4:2, 90-94 (2013); Ripka et al . Arch. Biochem. Biophys. 249:533-545 (1986); Yamane-Ohnuki et al . Biotech. Bioeng. 87: 614 (2004); Pereira et al . mAbs 10(5): 693-711 (2018).

在一些實施例中，本文所提供之FAP結合劑可進一步修飾以含有所屬技術領域種已知且容易獲得的額外非蛋白質部份。適合FAP結合劑衍生化的部份包括但不限於水溶性聚合物。水溶性聚合物的非限制性實例包括但不限於聚乙二醇(polyethylene glycol, PEG)、乙二醇/丙二醇的共聚物、羧甲基纖維素、右旋糖酐、聚乙烯醇、聚乙烯吡咯啶酮、聚-1,3-二氧戊環、聚-1,3,6-三㗁烷、乙烯/馬來酸酐共聚物、聚胺基酸（均聚物或隨機共聚物）、及右旋糖酐或聚(n-乙烯基吡咯啶酮)聚乙二醇、丙二醇均聚物、聚環氧丙烷/環氧乙烷共聚物、聚氧乙烯化多元醇（例如甘油）、聚乙烯醇、及其混合物。聚乙二醇丙醛因為其在水中的穩定性而在製造中可能具有優勢。聚合物可具有任何分子量，且可係分支的或非分支的。附接至抗體之聚合物的數目可變化，且若附接多於一種聚合物，則其等可係相同或不同的分子。通常，用於衍生化之聚合物的數目及/或類型可基於考慮因素來判定，包括但不限於待改進之FAP結合劑的特定性質或功能、抗體衍生物是否將在限定條件下用於治療。 活性 In some embodiments, the FAP binders provided herein can be further modified to include additional non-protein fractions known and readily available in the art. Suitable fractions for FAP binder derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-triane, ethylene/maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymers, polyoxyethyleneized polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer can have any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. Typically, the number and/or type of polymer used for derivatization can be determined based on factors including, but not limited to, the specific properties or functions of the FAP binder to be improved, and whether the antibody derivative will be used for treatment under specific conditions. Activity

在一些實施例中，本文所提供之FAP結合劑（例如抗FAP抗體或其抗原結合片段、雙特異性抗體、或其融合蛋白）可結合FAP。在一些實施例中，FAP結合劑（例如雙特異性抗體或其融合蛋白）可結合TACR。本文所提供之抗FAP抗體、雙特異性抗體、或其融合蛋白與目標（諸如FAP或TACR）之結合親和力可藉由測量例如與表現FAP之細胞系結合的半最大有效濃度(EC 50)來判定。以莫耳濃度（例如nM）表示的EC 50係達到最大結合的一半的抗FAP抗體、雙特異性抗體、或其融合蛋白的濃度。 In some embodiments, the FAP conjugates provided herein (e.g., anti-FAP antibodies or their antigen-binding fragments, bispecific antibodies, or their fusion proteins) can bind to FAP. In some embodiments, the FAP conjugates (e.g., bispecific antibodies or their fusion proteins) can bind to TACR. The binding affinity of the anti-FAP antibodies, bispecific antibodies, or their fusion proteins provided herein to a target (such as FAP or TACR) can be determined by measuring, for example, the half-maximal effective concentration ( EC50 ) of the anti-FAP antibody, bispecific antibody, or its fusion protein that binds to a cell line expressing FAP. The EC50, expressed in moles (e.g., nM), represents the concentration of the anti-FAP antibody, bispecific antibody, or its fusion protein at which half of the maximum binding is achieved.

在一些實施例中，本文所提供之抗FAP抗體、雙特異性抗體、或其融合蛋白可以不大於20 nM、不大於19 nM、不大於18 nM、不大於17 nM、不大於16 nM、不大於15 nM、不大於14 nM、不大於13 nM、不大於12 nM、不大於11 nM、不大於10 nM、不大於9 nM、不大於8 nM、不大於7 nM、不大於6 nM、或不大於5 nM的EC 50結合FAP或結合表現FAP之細胞。在一些實施例中，本文所提供之抗FAP抗體、雙特異性抗體、或其融合蛋白可以約0.001 nM至約5 nM、約0.01 nM至約3 nM、或約0.1 nM至約2 nM的EC 50結合FAP或結合表現FAP之細胞。 In some embodiments, the anti-FAP antibody, bispecific antibody, or fusion protein provided herein may be no more than 20 nM, no more than 19 nM, no more than 18 nM, no more than 17 nM, no more than 16 nM, no more than 15 nM, no more than 14 nM, no more than 13 nM, no more than 12 nM, no more than 11 nM, no more than 10 nM, no more than 9 nM, no more than 8 nM, no more than 7 nM, no more than 6 nM, or no more than 5 nM of EC50 binding to FAP or to cells expressing FAP. In some embodiments, the anti-FAP antibodies, bispecific antibodies, or fusion proteins thereof provided herein may bind to FAP or cells expressing FAP at EC50 concentrations of about 0.001 nM to about 5 nM, about 0.01 nM to about 3 nM, or about 0.1 nM to about 2 nM.

在一些實施例中，本文所提供之雙特異性抗體或其融合蛋白可以不大於20 nM、不大於19 nM、不大於18 nM、不大於17 nM、不大於16 nM、不大於15 nM、不大於14 nM、不大於13 nM、不大於12 nM、不大於11 nM、不大於10 nM、不大於9 nM、不大於8 nM、不大於7 nM、不大於6 nM、或不大於5 nM的EC 50結合TACR（例如LTßR）或結合表現TACR（例如LTßR）之細胞。在一些實施例中，本文所提供之雙特異性抗體或其融合蛋白可以約0.001 nM至約5 nM、約0.01 nM至約3 nM、或約0.1 nM至約2 nM的EC 50結合TACR（例如LTßR）或表現TACR（例如LTßR）之細胞。 In some embodiments, the bispecific antibody or its fusion protein provided herein may be in EC50 cells that bind TACR (e.g., LTßR) or bind cells that express TACR (e.g., LTßR) at a concentration not exceeding 20 nM, 19 nM, 18 nM, 17 nM, 16 nM, 15 nM, 14 nM, 13 nM, 12 nM, 11 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, or 5 nM. In some embodiments, the bispecific antibody or its fusion protein provided herein may be in EC50 cells that bind TACR (e.g., LTßR) or express TACR (e.g., LTßR) at a rate of about 0.001 nM to about 5 nM, about 0.01 nM to about 3 nM, or about 0.1 nM to about 2 nM.

本文所提供之抗FAP抗體、雙特異性抗體、或其融合蛋白與目標（諸如FAP或TACR（例如LTßR））之結合親和力可藉由測量例如解離常數（K D，K D=k off/k on，或K D= Kd/Ka）來判定。如本文中所使用，K D具有奈米莫耳單位(nM)且對應於在平衡時佔據一半目標蛋白的抗體、雙特異性抗體、或其融合蛋白的濃度。解離常數愈小，抗體、雙特異性抗體、或融合蛋白與其目標之間的親和力愈高。 The binding affinity of anti-FAP antibodies, bispecific antibodies, or their fusion proteins to targets (such as FAP or TACRs (e.g., LTßR)) presented in this article can be determined by measuring, for example, the dissociation constant (K <sub>D</sub> , K <sub>D</sub> = k<sub> off </sub>/k <sub>on</sub> , or K <sub>D</sub> = K<sub>d</sub>/Ka). As used herein, K <sub>D </sub> has nanomolar units (nM) and corresponds to the concentration of the antibody, bispecific antibody, or its fusion protein that constitutes half of the target protein at equilibrium. The smaller the dissociation constant, the higher the affinity between the antibody, bispecific antibody, or fusion protein and its target.

在一些實施例中，本文所提供之抗FAP抗體、雙特異性抗體、或融合蛋白可以不大於20 nM、不大於19 nM、不大於18 nM、不大於17 nM、不大於16 nM、不大於15 nM、不大於14 nM、不大於13 nM、不大於12 nM、不大於11 nM、不大於10 nM、不大於9 nM、不大於8 nM、不大於7 nM、不大於6 nM、或不大於5 nM的K D特異性結合FAP或特異性結合表現FAP之細胞。在一些實施例中，本文所提供之抗FAP抗體、雙特異性抗體、或融合蛋白可以約0.5 nM至約5 nM、約1 nM至約4 nM、或約2 nM至約3 nM的K D特異性結合FAP或特異性結合表現FAP之細胞。在一些實施例中，雙特異性抗體或融合蛋白以不大於20 nM、15 nM、10 nM、或5 nM之解離常數（KD，KD=koff/kon，或KD= Kd/Ka）結合至FAP。 In some embodiments, the anti-FAP antibody, bispecific antibody, or fusion protein provided herein may be no more than 20 nM, no more than 19 nM, no more than 18 nM, no more than 17 nM, no more than 16 nM, no more than 15 nM, no more than 14 nM, no more than 13 nM, no more than 12 nM, no more than 11 nM, no more than 10 nM, no more than 9 nM, no more than 8 nM, no more than 7 nM, no more than 6 nM, or no more than 5 nM KD- specifically binding to FAP or specifically binding to cells expressing FAP. In some embodiments, the anti-FAP antibodies, bispecific antibodies, or fusion proteins provided herein can specifically bind to FAP or cells expressing FAP at a KD of about 0.5 nM to about 5 nM, about 1 nM to about 4 nM, or about 2 nM to about 3 nM. In some embodiments, the bispecific antibodies or fusion proteins bind to FAP with a dissociation constant (KD, KD=koff/kon, or KD= Kd/Ka) not greater than 20 nM, 15 nM, 10 nM, or 5 nM.

在一些實施例中，本文所提供之雙特異性抗體或融合蛋白可以不大於20 nM、不大於19 nM、不大於18 nM、不大於17 nM、不大於16 nM、不大於15 nM、不大於14 nM、不大於13 nM、不大於12 nM、不大於11 nM、不大於10 nM、不大於9 nM、不大於8 nM、不大於7 nM、不大於6 nM、或不大於5 nM的K D特異性結合TACR（例如LTßR）或特異性結合表現TACR（例如LTßR）之細胞。在一些實施例中，本文所提供之雙特異性抗體或融合蛋白可以約0.1 nM至約10 nM、約0.2至約5 nM、或約0.3 nM至約3nM的K D特異性結合TACR（例如LTßR）或特異性表現TACR（例如LTßR）之細胞。在一些實施例中，雙特異性抗體或融合蛋白以不大於20 nM、15 nM、10 nM、或5 nM之解離常數(KD)結合TACR（例如LTßR）。 In some embodiments, the bispecific antibody or fusion protein provided herein may be no more than 20 nM, no more than 19 nM, no more than 18 nM, no more than 17 nM, no more than 16 nM, no more than 15 nM, no more than 14 nM, no more than 13 nM, no more than 12 nM, no more than 11 nM, no more than 10 nM, no more than 9 nM, no more than 8 nM, no more than 7 nM, no more than 6 nM, or no more than 5 nM of KD- specifically binding TACR (e.g., LTßR) or specifically binding to cells expressing TACR (e.g., LTßR). In some embodiments, the bispecific antibody or fusion protein provided herein can specifically bind to or specifically express a TACR (e.g., LTßR) in cells with a KD of about 0.1 nM to about 10 nM, about 0.2 nM to about 5 nM, or about 0.3 nM to about 3 nM. In some embodiments, the bispecific antibody or fusion protein binds to a TACR (e.g., LTßR) with a dissociation constant (KD) of no more than 20 nM, 15 nM, 10 nM, or 5 nM.

K D值可藉助於表面電漿子共振(surface plasmon resonance, SPR)光譜、生物層干涉法(biolayer interferometry, BLI)、或放射性標記抗原結合檢定(radiolabeled antigen-binding assay, RIA)來判定。K on、或結合速率、締合的速率、締合速率、以及k off、或脫離速率、解離的速率、解離速率亦可用上述相同的SPR或BLI技術來判定。當發現檢定條件會影響經判定K D，應使用標準偏差最小的檢定設置。 The K<sub>D</sub> value can be determined using surface plasmon resonance (SPR) spectroscopy, biolayer interferometry (BLI), or radiolabeled antigen-binding assay (RIA). K<sub>on </sub>, or binding rate, and k <sub>off </sub>, or dissociation rate, can also be determined using the same SPR or BLI techniques. When it is found that the test conditions affect the determined K<sub> D </sub>, the test settings with the smallest standard deviation should be used.

在一些實施例中，FAP結合劑（例如抗FAP抗體或其抗原結合片段、雙特異性抗體、或其融合蛋白）可特異性結合至FAP及/或TACR（例如以比與無關對照蛋白質（例如雞蛋清溶菌酶）或競爭分子之親和力大至少2、5、10、15、20、25、50、100、250、500、1000、10,000倍或更多倍的親和力。競爭分子可係非腫瘤相關分子、非腫瘤細胞表面分子、促腫瘤細胞表面分子（例如HVEM及/或DPPIV）、非人類細胞（例如食蟹獼猴HVEM）、及/或抑制劑分子（例如DcR3）。在一些實施例中，FAP結合劑（例如抗FAP抗體或其抗原結合片段、雙特異性抗體、或其融合蛋白）可與除人類FAP序列（例如食蟹獼猴序列）以外的FAP序列反應。例示性FAP及TACR序列分別描述於 表 5及 表 5.1中。 In some embodiments, FAP conjugates (e.g., anti-FAP antibodies or their antigen-binding fragments, bispecific antibodies, or their fusion proteins) can specifically bind to FAP and/or TACR (e.g., with an affinity at least 2, 5, 10, 15, 20, 25, 50, 100, 250, 500, 1000, 10,000 or more times greater than that with unrelated control proteins (e.g., egg white lysozyme) or competing molecules. Competing molecules can be non-tumor-related molecules or non-tumor cells. Surface molecules, tumor-promoting cell surface molecules (e.g., HVEM and/or DPPIV), non-human cells (e.g., cynomolgus monkey HVEM), and/or inhibitory molecules (e.g., DcR3). In some embodiments, FAP conjugates (e.g., anti-FAP antibodies or their antigen-binding fragments, bispecific antibodies, or their fusion proteins) may react with FAP sequences other than human FAP sequences (e.g., cynomolgus monkey sequences). Illustrative FAP and TACR sequences are described in Tables 5 and 5.1 , respectively.

在一些實施例中，FAP結合劑（例如抗FAP抗體或其抗原結合片段、雙特異性抗體、或其融合蛋白）相對於比較物結合劑展現出降低的與競爭性HVEM分子之結合親和力。在一些實施例中，相對於比較物結合劑，FAP結合劑（例如抗FAP抗體或其抗原結合片段、雙特異性抗體、或其融合蛋白）展現出與競爭性HVEM分子降低至少約1%、至少約5%、至少約10%、至少約20%、至少約30%、至少約40%、至少約50%、至少約60%、至少約70%、至少約80%、至少約90%、或100%之結合親和力。在一些實施例中，FAP結合劑（例如抗FAP抗體或其抗原結合片段、雙特異性抗體、或其融合蛋白）幾乎不結合至人類或食蟹獼猴HVEM。In some embodiments, the FAP conjugate (e.g., an anti-FAP antibody or its antigen-binding fragment, a bispecific antibody, or a fusion protein thereof) exhibits a reduced binding affinity to the competitive HVEM molecule relative to the comparison conjugate. In some embodiments, the FAP conjugate (e.g., an anti-FAP antibody or its antigen-binding fragment, a bispecific antibody, or a fusion protein thereof) exhibits a binding affinity to the competitive HVEM molecule that is at least about 1%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or 100% lower than the comparison conjugate. In some embodiments, FAP binders (such as anti-FAP antibodies or their antigen-binding fragments, bispecific antibodies, or their fusion proteins) hardly bind to human or cynomolgus monkey HVEMs.

在一些實施例中，相對於比較物結合劑，FAP結合劑（例如抗FAP抗體或其抗原結合片段、雙特異性抗體、或其融合蛋白）展現出與競爭性DcR3降低之結合親和力。在一些實施例中，相對於比較物結合劑，FAP結合劑（例如抗FAP抗體或其抗原結合片段、雙特異性抗體、或其融合蛋白）展現出與競爭性DcR3分子降低至少約1%、至少約5%、至少約10%、至少約20%、至少約30%、至少約40%、至少約50%、至少約60%、至少約70%、至少約80%、至少約90%、或100%之結合親和力。在一些實施例中，LIGHT突變體能夠降低與DcR3之結合親和力。In some embodiments, the FAP binder (e.g., an anti-FAP antibody or its antigen-binding fragment, a bispecific antibody, or a fusion protein thereof) exhibits a binding affinity for a competitive DcR3 reduction relative to the comparator. In some embodiments, the FAP binder (e.g., an anti-FAP antibody or its antigen-binding fragment, a bispecific antibody, or a fusion protein thereof) exhibits a binding affinity for a competitive DcR3 molecule that is at least about 1%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or 100% reduced. In some embodiments, the LIGHT mutant is able to reduce its binding affinity to DcR3.

在一些實施例中，相對於比較物結合劑，FAP結合劑（例如抗FAP抗體或其抗原結合片段、雙特異性抗體、或其融合蛋白）展現出與競爭性DPPIV降低之結合親和力。在一些實施例中，相對於比較物結合劑，FAP結合劑（例如抗FAP抗體或其抗原結合片段、雙特異性抗體、或其融合蛋白）展現出與競爭性DPPIV分子降低至少約1%、至少約5%、至少約10%、至少約20%、至少約30%、至少約40%、至少約50%、至少約60%、至少約70%、至少約80%、至少約90%、或100%之結合親和力。在一些實施例中，融合蛋白特異性結合至人類FAP，且/或不結合至DPPIV。In some embodiments, the FAP binder (e.g., an anti-FAP antibody or its antigen-binding fragment, a bispecific antibody, or a fusion protein thereof) exhibits a reduced binding affinity to the competitive DPPIV molecule relative to the comparison binder. In some embodiments, the FAP binder (e.g., an anti-FAP antibody or its antigen-binding fragment, a bispecific antibody, or a fusion protein thereof) exhibits a reduced binding affinity to the competitive DPPIV molecule of at least about 1%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or 100%. In some embodiments, the fusion protein specifically binds to human FAP and/or does not bind to DPPIV.

例示性的競爭性分子在實例中列出。Illustrative competitors are listed in the examples.

在一些實施例中，FAP結合劑（例如抗FAP抗體或其抗原結合片段、雙特異性抗體、或其融合蛋白）誘導樣本中之一或多種免疫細胞（例如樹突細胞、T細胞、及/或B細胞）的刺激。在一些實施例中，相對於比較物結合劑，FAP結合劑（例如抗FAP抗體或其抗原結合片段、雙特異性抗體、或其融合蛋白）誘導樣本中之一或多種免疫細胞（例如樹突細胞、T細胞、及/或B細胞）的刺激。在一些實施例中，相對於比較物結合劑，FAP結合劑（例如抗FAP抗體或其抗原結合片段、雙特異性抗體、或其融合蛋白）誘導樣本中至少約1%、至少約5%、至少約10%、至少約20%、至少約30%、至少約40%、至少約50%、至少約60%、至少約70%、至少約80%、至少約90%、或100%的免疫細胞（例如樹突細胞、T細胞、及/或B細胞）的刺激。In some embodiments, an FAP conjugate (e.g., an anti-FAP antibody or its antigen-binding fragment, a bispecific antibody, or its fusion protein) induces stimulation of one or more immune cells (e.g., dendritic cells, T cells, and/or B cells) in a sample. In some embodiments, the FAP conjugate (e.g., an anti-FAP antibody or its antigen-binding fragment, a bispecific antibody, or its fusion protein) induces stimulation of one or more immune cells (e.g., dendritic cells, T cells, and/or B cells) in a sample relative to a comparison conjugate. In some embodiments, the FAP binder (e.g., an anti-FAP antibody or its antigen-binding fragment, a bispecific antibody, or its fusion protein) induces stimulation of at least about 1%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or 100% of immune cells (e.g., dendritic cells, T cells, and/or B cells) in the sample relative to the comparison binder.

在一些實施例中，FAP結合劑（例如抗FAP抗體或其抗原結合片段、雙特異性抗體、或其融合蛋白）活化樣本中的一或多種癌相關纖維母細胞(cancer associated fibroblast cell, CAF)。在一些實施例中，相對於比較物結合劑，FAP結合劑（例如抗FAP抗體或其抗原結合片段、雙特異性抗體、或其融合蛋白）活化樣本中的一或多種CAF。在一些實施例中，相對於比較物結合劑，FAP結合劑（例如抗FAP抗體或其抗原結合片段、雙特異性抗體、或其融合蛋白物）活化樣本中至少約1%、至少約5%、至少約10%、至少約20%、至少約30%、至少約40%、至少約50%、至少約60%、至少約70%、至少約80%、至少約90%、或100%的CAF。In some embodiments, an FAP conjugate (e.g., an anti-FAP antibody or its antigen-binding fragment, a bispecific antibody, or a fusion protein thereof) activates one or more cancer-associated fibroblast cells (CAFs) in a sample. In some embodiments, the FAP conjugate (e.g., an anti-FAP antibody or its antigen-binding fragment, a bispecific antibody, or a fusion protein thereof) activates one or more CAFs in a sample relative to a comparison conjugate. In some embodiments, the FAP conjugate (e.g., an anti-FAP antibody or its antigen-binding fragment, a bispecific antibody, or its fusion protein) contains at least about 1%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or 100% CAF in the activated sample relative to the comparator conjugate.

在一些實施例中，FAP結合劑（例如抗FAP抗體或其抗原結合片段、雙特異性抗體、或其融合蛋白）影響次級淋巴器官(SLO)、三級淋巴結構(TLS)、或兩者的形成。在一些實施例中，相對於比較物結合劑，FAP結合劑（例如抗FAP抗體或其抗原結合片段、雙特異性抗體、或其融合蛋白）使得樣本中次級淋巴器官(SLO)、三級淋巴結構(TLS)、或兩者的形成增加。在一些實施例中，相對於比較物結合劑，FAP結合劑（例如抗FAP抗體或其抗原結合片段、雙特異性抗體、或其融合蛋白）使得樣本中次級淋巴器官(SLO)、三級淋巴結構(TLS)、或兩者的形成增加至少約1%、至少約5%、至少約10%、至少約20%、至少約30%、至少約40%、至少約50%、至少約60%、至少約70%、至少約80%、至少約90%、至少約100%、至少約110%、至少約120%、至少約130%、至少約140%、至少約150%、至少約160%、至少約170%、至少約180%、至少約190%、或至少約200%。In some embodiments, FAP conjugates (e.g., anti-FAP antibodies or their antigen-binding fragments, bispecific antibodies, or their fusion proteins) affect the formation of secondary lymphoid organs (SLOs), tertiary lymphoid structures (TLSs), or both. In some embodiments, FAP conjugates (e.g., anti-FAP antibodies or their antigen-binding fragments, bispecific antibodies, or their fusion proteins) increase the formation of secondary lymphoid organs (SLOs), tertiary lymphoid structures (TLSs), or both in a sample, relative to a comparison conjugate. In some embodiments, the FAP binder (e.g., an anti-FAP antibody or its antigen-binding fragment, a bispecific antibody, or its fusion protein) increases the formation of secondary lymphoid organs (SLO), tertiary lymphoid structures (TLS), or both in a sample by at least about 1%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, or at least about 200%, relative to the comparator binder.

在一些實施例中，相對於比較物結合劑，FAP結合劑（例如抗FAP抗體或其抗原結合片段、雙特異性抗體、或其融合蛋白）使得樣本中腫瘤生長速率或腫瘤細胞數目降低。在一些實施例中，相對於比較物結合劑，FAP結合劑（例如抗FAP抗體或其抗原結合片段、雙特異性抗體、或其融合蛋白）使得樣本中腫瘤生長速率或腫瘤細胞數目降低至少約1%、至少約5%、至少約10%、至少約20%、至少約30%、至少約40%、至少約50%、至少約60%、至少約70%、至少約80%、至少約90%、或100%。In some embodiments, the FAP conjugate (e.g., an anti-FAP antibody or its antigen-binding fragment, a bispecific antibody, or a fusion protein thereof) reduces the tumor growth rate or the number of tumor cells in the sample relative to the comparison conjugate. In some embodiments, the FAP conjugate (e.g., an anti-FAP antibody or its antigen-binding fragment, a bispecific antibody, or a fusion protein thereof) reduces the tumor growth rate or the number of tumor cells in the sample by at least about 1%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or 100% relative to the comparison conjugate.

在一些實施例中，相對於比較物結合劑，FAP結合劑（例如抗FAP抗體或其抗原結合片段、雙特異性抗體、或其融合蛋白）使得樣本中腫瘤細胞死亡增加。在一些實施例中，相對於比較物結合劑，FAP結合劑（例如抗FAP抗體或其抗原結合片段、雙特異性抗體、或其融合蛋白）使得樣本中腫瘤細胞死亡增加至少約1%、至少約5%、至少約10%、至少約20%、至少約30%、至少約40%、至少約50%、至少約60%、至少約70%、至少約80%、至少約90%、至少約100%、至少約110%、至少約120%、至少約130%、至少約140%、至少約150%、至少約160%、至少約170%、至少約180%、至少約190%、或至少約200%。 產生及表現FAP 結合劑多核苷酸 In some embodiments, FAP binders (such as anti-FAP antibodies or their antigen-binding fragments, bispecific antibodies, or their fusion proteins) increase tumor cell death in samples compared to comparator binders. In some embodiments, the FAP conjugate (e.g., an anti-FAP antibody or its antigen-binding fragment, a bispecific antibody, or its fusion protein) increases tumor cell death in the sample by at least about 1%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, or at least about 200%, relative to the comparison conjugate. The FAP conjugate polynucleotide is generated and expressed.

本揭露提供編碼FAP結合劑（例如抗FAP抗體或其抗原結合片段、包含其的雙特異性抗體、或上述融合蛋白）或其部分的一或多種經單離多核苷酸。多核苷酸係DNA、RNA、DNA/RNA雜合體的聚合物、或其修飾。在一些實施例中，多核苷酸係DNA的聚合物。在一些實施例中，多核苷酸係RNA的聚合物。This disclosure provides one or more isolated polynucleotides encoding FAP binding agents (e.g., anti-FAP antibodies or antigen-binding fragments thereof, bispecific antibodies containing thereof, or fusion proteins thereof). The polynucleotide is a polymer of DNA, RNA, a DNA/RNA hybrid, or a modification thereof. In some embodiments, the polynucleotide is a polymer of DNA. In some embodiments, the polynucleotide is a polymer of RNA.

本文亦提供包含編碼FAP結合劑（例如抗FAP抗體或其抗原結合片段、包含其的雙特異性抗體、或上述融合蛋白）或其部分的至少一個多核苷酸的核酸分子。在一些實施例中，核酸分子包含編碼FAP結合劑的重鏈或輕鏈的多核苷酸。在一些實施例中，核酸分子包含編碼FAP結合劑的第一重鏈的多核苷酸、編碼第二重鏈的多核苷酸、及編碼輕鏈的多核苷酸。在一些實施例中，第一核酸分子包含編碼重鏈的第一多核苷酸且第二核酸分子包含編碼輕鏈的第二多核苷酸。在一些實施例中，第一核酸分子包含編碼重鏈的第一多核苷酸，第二核酸分子包含編碼輕鏈的第二多核苷酸，且第三核酸分子包含編碼第二重鏈的第三多核苷酸。在一些實施例中，重鏈及輕鏈從一個核酸分子表現，或從兩個單獨的核酸分子表現為兩個單獨的多肽。在一些實施例中，第一重鏈、第二重鏈、及輕鏈從一個核酸分子表現、從兩個單獨的核酸分子表現為兩個單獨的多肽、或從三個單獨的核酸分子表現為三個單獨的多肽。在一些實施例中，單一多核苷酸編碼包含連接在一起的第一重鏈、第二重鏈、及輕鏈的單一多肽。This document also provides nucleic acid molecules comprising at least one polynucleotide encoding an FAP conjugate (e.g., an anti-FAP antibody or its antigen-binding fragment, a bispecific antibody containing the same, or a fusion protein thereof). In some embodiments, the nucleic acid molecule comprises a polynucleotide encoding a heavy chain or a light chain of the FAP conjugate. In some embodiments, the nucleic acid molecule comprises a polynucleotide encoding a first heavy chain, a polynucleotide encoding a second heavy chain, and a polynucleotide encoding a light chain. In some embodiments, the first nucleic acid molecule comprises a first polynucleotide encoding a heavy chain and the second nucleic acid molecule comprises a second polynucleotide encoding a light chain. In some embodiments, the first nucleic acid molecule comprises a first polynucleotide encoding a heavy chain, the second nucleic acid molecule comprises a second polynucleotide encoding a light chain, and the third nucleic acid molecule comprises a third polynucleotide encoding a second heavy chain. In some embodiments, the heavy chain and light chain are expressed from a single nucleic acid molecule, or from two separate nucleic acid molecules as two separate polypeptides. In some embodiments, the first heavy chain, second heavy chain, and light chain are expressed from a single nucleic acid molecule, from two separate nucleic acid molecules as two separate polypeptides, or from three separate nucleic acid molecules as three separate polypeptides. In some embodiments, a single polynucleotide encodes a single polypeptide comprising a first heavy chain, a second heavy chain, and a light chain linked together.

在一些實施例中，當抗體係雙特異性抗體時，核苷酸分子包含編碼第一靶向部份（例如抗FAP抗體或其抗原結合片段）的多核苷酸及編碼第二靶向部份（例如第二抗體或其抗原結合片段或TACR）的多核苷酸兩者。在一些實施例中，第一核酸分子包含編碼第一靶向部份（例如抗FAP抗體或其抗原結合片段）的第一多核苷酸，且第二核酸分子包含編碼第二靶向部份（例如第二抗體或其抗原結合片段或TACR）的第二多核苷酸。在一些實施例中，第一靶向部份（例如抗FAP抗體或其抗原結合片段）及第二靶向部份（例如第二抗體或其抗原結合片段或TACR）從一個核酸分子表現或從兩個單獨的核酸分子表現為兩個單獨的多肽。在一些實施例中，單一多核苷酸編碼包含連接在一起的第一靶向部份（例如抗FAP抗體或其抗原結合片段）及第二靶向部份（例如第二抗體或其抗原結合片段或TACR）兩者的單一多肽。In some embodiments, when the antibody system is a bispecific antibody, the nucleotide molecule comprises both a polynucleotide encoding a first targeting portion (e.g., an anti-FAP antibody or its antigen-binding fragment) and a polynucleotide encoding a second targeting portion (e.g., a second antibody or its antigen-binding fragment or TACR). In some embodiments, the first nucleic acid molecule comprises a first polynucleotide encoding the first targeting portion (e.g., an anti-FAP antibody or its antigen-binding fragment), and the second nucleic acid molecule comprises a second polynucleotide encoding the second targeting portion (e.g., a second antibody or its antigen-binding fragment or TACR). In some embodiments, the first targeting portion (e.g., an anti-FAP antibody or its antigen-binding fragment) and the second targeting portion (e.g., a second antibody or its antigen-binding fragment or TACR) are expressed from a single nucleic acid molecule or from two separate nucleic acid molecules as two separate polypeptides. In some embodiments, a single polynucleotide encoding a single polypeptide comprising a first targeting portion (e.g., an anti-FAP antibody or its antigen-binding fragment) and a second targeting portion (e.g., a second antibody or its antigen-binding fragment or TACR) linked together.

所屬技術領域中具有通常知識者將理解，本文所述之任何單一多核苷酸或多核苷酸的組合可包含在單一核酸分子上或多於一個核酸分子上，諸如在單獨的核酸分子上。Those skilled in the art will understand that any single polynucleotide or combination of polynucleotides described herein may be contained on a single nucleic acid molecule or on more than one nucleic acid molecule, such as on a single nucleic acid molecule.

在一些實施例中，編碼FAP結合劑的重鏈或輕鏈的多核苷酸包含編碼本文所提供之CDR中的至少一者的核苷酸序列。在一些實施例中，編碼FAP結合劑的重鏈或輕鏈的多核苷酸包含編碼本文所提供之CDR中的至少3者的核苷酸序列。在一些實施例中，編碼FAP結合劑的重鏈或輕鏈的多核苷酸包含編碼本文所提供之CDR中的至少6者的核苷酸序列。在一些實施例中，編碼FAP結合劑的重鏈或輕鏈的多核苷酸包含編碼前導序列的核苷酸序列，該前導序列在轉譯時位於重鏈或輕鏈之N端。如上所述，前導序列可係天然重鏈或輕鏈前導序列，或可係另一異源前導序列。In some embodiments, the polynucleotide encoding the heavy or light chain of the FAP conjugate comprises a nucleotide sequence encoding at least one of the CDRs provided herein. In some embodiments, the polynucleotide encoding the heavy or light chain of the FAP conjugate comprises a nucleotide sequence encoding at least three of the CDRs provided herein. In some embodiments, the polynucleotide encoding the heavy or light chain of the FAP conjugate comprises a nucleotide sequence encoding at least six of the CDRs provided herein. In some embodiments, the polynucleotide encoding the heavy or light chain of the FAP conjugate comprises a nucleotide sequence encoding a leader sequence located at the N-terminus of the heavy or light chain during transcription. As described above, the leader sequence may be a natural heavy or light chain leader sequence or another heterologous leader sequence.

在一些實施例中，多核苷酸係編碼本文所提供之FAP結合劑之胺基酸序列中之任一者的多核苷酸。在一些實施例中，多核苷酸係與編碼本文所述之 表 6 至表10中之胺基酸序列中之任一者的核苷酸序列至少80%相同的多核苷酸，例如至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、或100%相同的多核苷酸。 In some embodiments, the polynucleotide is a polynucleotide that encodes any of the amino acid sequences of the FAP binders provided herein. In some embodiments, the polynucleotide is a polynucleotide whose nucleotide sequence is at least 80% identical to that of any of the amino acid sequences in Tables 6 to 10 described herein, for example, at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical.

在一些實施例中，本文所提供之多核苷酸包含編碼如本文所述之FAP結合劑（例如抗FAP抗體或其抗原結合片段、包含其的雙特異性抗體、或上述融合蛋白）或其部分（例如域、區、或部份）的核苷酸序列。在一些實施例中，本文所提供之多核苷酸包含編碼 表 6 至表10所述之胺基酸序列中之任一或多者的核苷酸序列。在一些實施例中，本文所提供之多核苷酸包含編碼 表 6 或表7所述之CDR序列中之任一或多者的核苷酸序列。在一些實施例中，本文所提供之多核苷酸包含編碼 表 8所述之VH及/或VL序列中之任一或多者的核苷酸序列。在一些實施例中，本文所提供之多核苷酸包含編碼 表 9所述之HC及/或LC序列中之任一或多者的核苷酸序列。在一些實施例中，本文所提供之多核苷酸包含編碼 表 10所述之胺基酸序列中之任一或多者的核苷酸序列。 In some embodiments, the polynucleotides provided herein comprise nucleotide sequences encoding FAP binders as described herein (e.g., anti-FAP antibodies or their antigen-binding fragments, bispecific antibodies comprising them, or the aforementioned fusion proteins) or portions thereof (e.g., domains, regions, or parts). In some embodiments, the polynucleotides provided herein comprise nucleotide sequences encoding any or more of the amino acid sequences described in Tables 6 to 10. In some embodiments, the polynucleotides provided herein comprise nucleotide sequences encoding any or more of the CDR sequences described in Tables 6 or 7. In some embodiments, the polynucleotides provided herein comprise nucleotide sequences encoding any or more of the VH and/or VL sequences described in Table 8. In some embodiments, the polynucleotides provided herein comprise nucleotide sequences encoding any or more of the HC and/or LC sequences described in Table 9 . In some embodiments, the polynucleotides provided herein comprise nucleotide sequences of any or more of the amino acid sequences described in Table 10 .

在一些實施例中，多核苷酸係與本文所提供之多核苷酸序列中之任一或多者雜交的多核苷酸。在一些實施例中，雜交在中等條件下進行。在一些實施例中，雜交在高度嚴格的條件下進行，諸如：在65℃下至少約6X SSC及1% SDS，第一次在約42℃下用0.1X SSC中的約20% (v/v)甲醯胺洗滌10分鐘，且隨後在65℃下用0.2 X SSC及0.1% SDS洗滌。In some embodiments, the polynucleotide is a polynucleotide hybridized with one or more of the polynucleotide sequences provided herein. In some embodiments, hybridization is performed under moderate conditions. In some embodiments, hybridization is performed under highly stringent conditions, such as: at least about 6X SSC and 1% SDS at 65°C, a first wash at about 42°C with about 20% (v/v) methylamine in 0.1X SSC for 10 minutes, followed by a wash at 65°C with 0.2X SSC and 0.1% SDS.

編碼FAP結合劑（例如抗FAP抗體或其抗原結合片段、包含其的雙特異性抗體、或上述融合蛋白）或其部分的DNA或RNA易使用習知程序（例如藉由使用能夠特異性結合編碼抗體之重鏈及輕鏈之基因的寡核苷酸探針）單離及定序。編碼DNA或RNA亦可由合成方法獲得。DNA or RNA encoding FAP binding agents (e.g., anti-FAP antibodies or their antigen-binding fragments, bispecific antibodies containing them, or the aforementioned fusion proteins) or portions thereof can be isolated and sequenced using learned procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of antibodies). The encoding DNA or RNA can also be obtained synthetically.

可使用所屬技術領域中已知的重組技術將經單離多核苷酸插入構築體中以用於進一步選殖（DNA的擴增）或用於表現。Recombination techniques known in the relevant technical field can be used to insert isolated polynucleotides into the building blocks for further selection (DNA amplification) or for expression.

許多構築體係可用的。構築體組分通常包括但不限於以下中的一或多者：信號序列、複製起點、一或多個標記基因、增強要素、啟動子（例如SV40、CMV、EF-1α）、及轉錄終止序列。 構築體及載體 Many building blocks are available. Building block components typically include, but are not limited to, one or more of the following: a signaling sequence, a replication origin, one or more marker genes, enhancing elements, a promoter (e.g., SV40, CMV, EF-1α), and a transcription termination sequence. Buildings and Vectors

本文提供一種構築體，其包括上文提供之經單離多核苷酸。建構構築體的方法係所屬技術領域中具有通常知識者已知的。例如，構築體可由體外重組DNA技術、DNA合成技術、或體外重組技術獲得。更具體而言，可藉由將經單離多核苷酸插入表現載體的多株位點來建構。本揭露中的表現載體通常係指所屬技術領域中熟知的各種市售表現載體，例如細菌質體、噬菌體、酵母菌質體、感染植物細胞的病毒、諸如腺病毒、反轉錄病毒的感染哺乳動物細胞的病毒、或其他載體。This document provides a structure comprising the isolated polynucleotide described above. Methods for constructing the structure are known to those skilled in the art. For example, the structure can be obtained using in vitro recombinant DNA technology, DNA synthesis technology, or in vitro recombination technology. More specifically, it can be constructed by inserting the isolated polynucleotide into multiple sites of a expression vector. The expression vectors disclosed herein generally refer to various commercially available expression vectors well known in the art, such as bacterial plasmids, bacteriophages, yeast plasmids, viruses that infect plant cells, adenoviruses, retrotransmitters that infect mammalian cells, or other vectors.

在一些實施例中，載體可包括本文所述之多核苷酸中之任一或多者。例如，載體可包括編碼FAP結合劑（例如抗FAP抗體或其抗原結合片段、包含其的雙特異性、或上述融合蛋白）的多核苷酸，或多於一個載體可包括FAP結合劑之組分的任何組合（例如抗FAP抗體或其抗原結合片段、包含其的雙特異性、或上述融合蛋白、重鏈、可選地第二重鏈、輕鏈、第一靶向部分、或第二靶向部分、或其任何組合）。In some embodiments, the vector may include any or more of the polynucleotides described herein. For example, the vector may include a polynucleotide encoding an FAP conjugate (e.g., an anti-FAP antibody or its antigen-binding fragment, including its bispecificity, or the aforementioned fusion protein), or more than one vector may include any combination of components of an FAP conjugate (e.g., an anti-FAP antibody or its antigen-binding fragment, including its bispecificity, or the aforementioned fusion protein, a heavy chain, optionally a second heavy chain, a light chain, a first targeting portion, or a second targeting portion, or any combination thereof).

載體亦可包括可操作地連接至多核苷酸序列的一或多個調控序列，其中調控序列可包括適合的啟動子序列。啟動子序列通常可操作地連接至寫碼待表現的胺基酸序列的序列。啟動子可係在選定宿主細胞中表現出轉錄活性的任何核苷酸序列，包括突變的、截短的、及雜合的啟動子，且可從編碼與宿主細胞同源或異源的胞外或胞內多肽的基因獲得。The vector may also include one or more regulatory sequences operatively linked to a polynucleotide sequence, wherein the regulatory sequence may include a suitable promoter sequence. The promoter sequence is typically operatively linked to a sequence that encodes an amino acid sequence to be expressed. The promoter can be any nucleotide sequence that expresses transcriptional activity in a selected host cell, including mutated, truncated, and hybrid promoters, and can be obtained from a gene encoding an extracellular or intracellular polypeptide that is homologous or heterologous to the host cell.

調控序列可進一步包括適合的轉錄終止子序列，亦即由宿主細胞識別以終止轉錄的序列。終止子序列連接至編碼多肽的核苷酸序列的3’端或末端，且在選擇的宿主細胞中有功能的任何終止子均可用於本揭露。The regulatory sequence may further include a suitable transcriptional terminator sequence, i.e. a sequence that is recognized by the host cell to terminate transcription. The terminator sequence is attached to the 3' end or end of the nucleotide sequence encoding the polypeptide, and any terminator that is functional in the selected host cell may be used in this disclosure.

通常，適合的載體可含有能夠在至少一個生物體中複製的起點、啟動子序列、方便的限制性酵素位點、及一或多個可選標記。例如，這些啟動子可包括但不限於大腸桿菌(E. coli)的lac或trp啟動子；λ噬菌體PL啟動子；及真核啟動子（包括CMV立即早期啟動子、HSV胸苷激酶啟動子、SV40早期及晚期啟動子、畢赤酵母甲醇氧化酶啟動子）、及能夠控制原核細胞或真核細胞或病毒中基因表現的一些其他已知啟動子。Typically, a suitable vector may contain an origin of replication in at least one organism, a promoter sequence, a convenient restriction enzyme site, and one or more optional markers. For example, these promoters may include, but are not limited to, the lac or trp promoter of E. coli; the PL promoter of λ phage; and eukaryotic promoters (including the CMV immediate early promoter, the HSV thymidine kinase promoter, the SV40 early and late promoters, and the Pichia pastoris methanol oxidase promoter), as well as some other known promoters that can control gene expression in prokaryotic or eukaryotic cells or viruses.

標記基因或可選擇標記可用以提供用於選擇經轉化宿主細胞的表型特徵。例如，標記基因可包括但不限於用於真核細胞培養物的二氫葉酸還原酶、新黴素抗性及綠螢光蛋白(green fluorescent protein, GFP)、或用於大腸桿菌的四環素抗性或安比西林抗性。Marker genes or optional markers can be used to provide phenotypic characteristics for selecting transformed host cells. For example, marker genes may include, but are not limited to, dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell cultures, or tetracycline resistance or ampicillin resistance for E. coli.

當表現多核苷酸時，表現載體可進一步包括增強子序列。若將增強子序列插入載體中，轉錄將會增強。增強子係DNA的順式作用因子，一般含有約10至300個鹼基對。增強子作用於啟動子以增強基因轉錄。When expressing polynucleotides, the expression vector may further include enhancer sequences. Inserting an enhancer sequence into the vector will enhance transcription. Enhancers are cis-acting factors of DNA and typically contain approximately 10 to 300 base pairs. Enhancers act on the promoter to enhance gene transcription.

若為所欲，一或多種多核苷酸亦可選地包含編碼與多肽序列框內融合的分泌信號肽的核苷酸序列。分泌信號肽引導表現一或多種核酸的細胞分泌抗體多肽，且由細胞從分泌的多肽中裂解。一或多種核酸可進一步可選地包含其唯一預期功能係促進載體的大規模生產的序列。可使用文獻中描述的用於多種轉殖基因的程序來製造及投予用於基因療法的核酸。參見例如Isner et al., Circulation, 91: 2687-2692 (1995)；及Isner et al., Human Gene Therapy, 7: 989-1011 (1996)。 If desired, one or more polynucleotides may also optionally include a nucleotide sequence encoding a secretory signaling peptide fused within the polypeptide sequence frame. The secretory signaling peptide induces the cell expressing one or more nucleic acids to secrete an antibody polypeptide, which is then cleaved by the cell from the secreted polypeptide. One or more nucleic acids may further optionally include a sequence whose sole intended function is to facilitate the large-scale production of the vector. Nucleic acids for gene therapy can be manufactured and delivered using procedures described in the literature for various transgenic genes. See, for example, Isner et al ., Circulation, 91: 2687-2692 (1995); and Isner et al ., Human Gene Therapy, 7: 989-1011 (1996).

在一些實施例中，一或多種多核苷酸可進一步包含額外的序列以促進宿主細胞的攝取及抗體或其片段（及/或任何其他肽）的表現。在一些實施例中，採用編碼本文所述之FAP結合劑或其部分的「裸露」轉殖基因（例如沒有病毒、微脂體、或其他載體以促進轉染的轉殖基因）。In some embodiments, one or more polynucleotides may further include additional sequences to promote uptake by host cells and expression of the antibody or fragment thereof (and/or any other peptide). In some embodiments, a "naked" transgenic gene (e.g., a transgenic gene without a virus, liposome, or other vector to promote transfection) encoding the FAP binder described herein or a portion thereof is used.

任何適合的載體可用以將編碼FAP結合劑的一或多種多核苷酸引入宿主中。已描述的例示性載體包括複製缺陷型反轉錄病毒載體，包括但不限於慢病毒載體（參見例如Kim et al., J. Virol., 72(1): 811-816 (1998)；Kingsman & Johnson, Scrip Magazine, October, 1998, pp. 43-46）；小病毒載體，諸如腺相關病毒(adeno-associated viral, AAV)載體（美國專利第5,474,935l號；第5,139,941號；第5,622,856號；第5,658,776號；第5,773,289號；第5,789,390號；第5,834,441號；第5,863,541號；第5,851,521號；第5,252,479號；Gnatenko et al., J. Invest. Med., 45: 87-98, (1997)）；腺病毒(adenoviral, AV)載體（參見例如美國專利第5,792,453號；第5,824,544第；第5,707,618第；第5,693,509第；第5,670,488第；第5,585,362第；Quantin et al., Proc. Natl. Acad. Sci. USA, 89: 2581-2584 (1992)；Stratford Perricaudet et al., J. Clin. Invest., 90: 626-630 (1992)；及Rosenfeld et al., Cell, 68: 143-155, (1992)）；腺病毒腺相關病毒嵌合體（美國專利第5,856,152號）或痘瘡病毒或疱疹病毒載體（美國專利第5,879,934號；第5,849,571號；第5,830,727號；第5,661,033號；第5328688號）；脂質體(Lipofectin)介導之基因轉移(BRL)；微脂體載體（美國專利第5,631,237號）；及其組合。可選地，藉由例如缺失或破壞病毒複製所需的選擇基因使病毒載體具有複製缺陷。 Any suitable vector can be used to introduce one or more polynucleotides encoding the FAP binder into the host. Exemplary vectors described include replication-defective retroviral vectors, including but not limited to lentiviral vectors (see, for example, Kim et al ., J. Virol., 72(1): 811-816 (1998); Kingsman & Johnson, Scrip Magazine, October, 1998, pp. 43-46); and microviral vectors, such as adeno-associated viruses. AAV vectors (US Patent Nos. 5,474,9351; 5,139,941; 5,622,856; 5,658,776; 5,773,289; 5,789,390; 5,834,441; 5,863,541; 5,851,521; 5,252,479; Gnatenko et al ., J. Invest. Med., 45: 87-98, (1997)); adenovirus (adenoviral, AV) carriers (see, for example, U.S. Patents 5,792,453; 5,824,544; 5,707,618; 5,693,509; 5,670,488; 5,585,362; Quantin et al ., Proc. Natl. Acad. Sci. USA, 89: 2581-2584 (1992); Stratford Perricaudet et al ., J. Clin. Invest., 90: 626-630 (1992); and Rosenfeld et al ., Cell, 68: 143-155, (1992)); adenovirus-associated virus chimeras (US Patent No. 5,856,152) or vaccinia virus or herpesvirus vectors (US Patent Nos. 5,879,934; 5,849,571; 5,830,727; 5,661,033; 5328688); liposome-mediated gene transfer (BRL); microliposome vectors (US Patent No. 5,631,237); and combinations thereof. Alternatively, the viral vector may be made replication-defective by, for example, deleting or disrupting a selectable gene required for viral replication.

此等表現載體中之任一者可使用描述於例如下列中之標準重組DNA技術製備：Sambrook et al., Molecular Cloning, a Laboratory Manual, 2d edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989)及Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York, N.Y. (1994)。 Any of these expression vectors can be prepared using standard recombinant DNA techniques described, for example, in: Sambrook et al ., Molecular Cloning, a Laboratory Manual, 2d edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989) and Ausubel et al ., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York, NY (1994).

預期的其他非病毒遞送機制包括磷酸鈣沉澱(Graham and Van Der Eb, Virology, 52: 456-467 (1973); Chen and Okayama, Mol. Cell Biol., 7: 2745-2752, 1987; Rippe et al., Mol. Cell Biol., 10: 689-695 (1990))、DEAE-右旋糖酐(Gopal, Mol. Cell Biol., 5: 1188-1190, (1985))、電穿孔(Tur-Kaspa et al., Mol. Cell Biol., 6: 716-718 (1986); Potter et al., Proc. Nat. Acad. Sci. USA, 81: 7161-7165 (1984))、直接顯微注射(Harland and Weintraub, J. Cell Biol., 101: 1094-1099 (1985))、載DNA之微脂體(DNA-loaded liposome) (Nicolau and Sene, Biochim. Biophys. Acta, 721: 185-190 (1982); Fraley et al., Proc. Natl. Acad. Sci. USA, 76: 3348-3352 (1979); Felgner, Sci Am., 276(6): 102-6, 1997; Felgner, Hum Gene Ther., 7(15): 1791-3 (1996))、細胞超音波處理(Fechheimer et al., Proc. Natl. Acad. Sci. USA, 84: 8463-8467 (1987))、使用高速微彈的基因轟擊(Yang et al., Proc. Natl. Acad. Sci USA, 87: 9568-9572 (1990))、及受體介導的轉染(Wu and Wu, J. Biol. Chem., 262: 4429-4432 (1987); Wu and Wu, Biochemistry, 27: 887-892 (1988); Wu and Wu, Adv. Drug Delivery Rev., 12: 159-167 (1993))。 Other anticipated non-viral delivery mechanisms include calcium phosphate precipitation (Graham and Van Der Eb, Virology, 52: 456-467 (1973); Chen and Okayama, Mol. Cell Biol., 7: 2745-2752, 1987; Rippe et al ., Mol. Cell Biol., 10: 689-695 (1990)), DEAE-dextran (Gopal, Mol. Cell Biol., 5: 1188-1190, (1985)), electroporation (Tur-Kaspa et al ., Mol. Cell Biol., 6: 716-718 (1986); Potter et al ., Proc. Nat. Acad. Sci. USA, 81: 7161-7165 (1984)), and direct microinjection (Harland and Weintraub, J. Cell Biol., 101: 1094-1099 (1985)), DNA-loaded liposomes (Nicolau and Sene, Biochim. Biophys. Acta, 721: 185-190 (1982); Fraley et al ., Proc. Natl. Acad. Sci. USA, 76: 3348-3352 (1979); Felgner, Sci Am., 276(6): 102-6, 1997; Felgner, Hum Gene Ther., 7(15): 1791-3 (1996)), and cellular ultrasound treatment (Fechheimer et al ., Proc. Natl. Acad. Sci. USA, 84: 8463-8467). (1987)), gene bombardment using high-speed microbombs (Yang et al ., Proc. Natl. Acad. Sci USA, 87: 9568-9572 (1990)), and receptor-mediated transfection (Wu and Wu, J. Biol. Chem., 262: 4429-4432 (1987); Wu and Wu, Biochemistry, 27: 887-892 (1988); Wu and Wu, Adv. Drug Delivery Rev., 12: 159-167 (1993)).

表現載體（或本文所述之抗體或其片段）可截留於微脂體中。參見例如Ghosh and Bachhawat, In: Liver diseases, targeted diagnosis and therapy using specific receptors and ligands, Wu G, Wu C ed., New York: Marcel Dekker, pp. 87-104 (1991)；Radler et al., Science, 275(5301): 810-814 (1997)。亦考慮了涉及「脂轉染(lipofection)」技術的各種商用方法。在一些實施例中，微脂體可與血球凝集病毒(hemagglutinating virus, HVJ)錯合。此已被顯示可促進與細胞膜的融合且促進微脂體囊封的DNA進入細胞（參見例如Kaneda et al., Science, 243: 375-378 (1989)）。在一些實施例中，微脂體與核非組蛋白染色體蛋白(HMG-1)錯合或結合使用（參見例如Kato et al., J. Biol. Chem., 266: 3361-3364 (1991)）。在一些實施例中，微脂體與HVJ及HMG-1兩者錯合或結合使用。此類表現構築體已成功用於核酸的體外及體內轉移及表現。在一些實施例中，包括人類FAP結合劑的FAP結合劑（例如抗體）包括在微脂體中以將微脂體靶向在其表面上表現FAP之細胞（諸如腫瘤細胞）。 抗體表現系統 The expression vector (or the antibody or its fragment described herein) can be trapped in liposomes. See, for example, Ghosh and Bachhawat, In: Liver diseases, targeted diagnosis and therapy using specific receptors and ligands, Wu G, Wu C ed., New York: Marcel Dekker, pp. 87-104 (1991); Radler et al ., Science, 275(5301): 810-814 (1997). Various commercial methods involving lipofection techniques are also considered. In some embodiments, liposomes can mismatch with hemagglutinating virus (HVJ). This has been shown to promote fusion with the cell membrane and facilitate the entry of DNA encapsulated in liposomes into cells (see, for example, Kaneda et al ., Science, 243: 375-378 (1989)). In some embodiments, liposomes are used in combination with or bound to nuclear non-histone chromosomal protein (HMG-1) (see, for example, Kato et al ., J. Biol. Chem., 266: 3361-3364 (1991)). In some embodiments, liposomes are used in combination with or bound to both HVJ and HMG-1. These performance constructs have been successfully used for the in vitro and in vivo transfer and expression of nucleic acids. In some embodiments, FAP-binding agents (e.g., antibodies), including human FAP-binding agents, are contained in liposomes to target the liposomes to cells (such as tumor cells) that express FAP on their surface. Antibody expression system.

本揭露提供一種抗體表現系統，其包括上文提供之構築體或將上文提供之外源多核苷酸或上文提供之多於一種外源多核苷酸併入細胞的基因體中。因此，本文提供一種細胞，諸如宿主細胞，或其用途。This disclosure provides an antibody expression system comprising the constructs provided above or incorporating the exogenous polynucleotides provided above, or more than one exogenous polynucleotide provided above, into a cell's genome. Therefore, this document provides a cell, such as a host cell, or its use.

本文所述之細胞，且具體地本文所述之宿主細胞可指用以接受、維持、複製、及擴增本文所提供之載體的細胞。宿主細胞亦可用以包含本文所提供之FAP結合劑，或表現由載體編碼的本文所提供之FAP結合劑（例如抗FAP抗體或其抗原結合片段、包含其的雙特異性抗體、或上述融合蛋白）。載體中包含的多核苷酸在宿主細胞分裂時複製，藉以擴增本文中包含的核酸。The term "cell" as used herein, and more specifically, "host cell" as used herein, can refer to a cell used to receive, maintain, replicate, and amplify the vectors provided herein. The host cell may also be used to contain the FAP binder provided herein, or to express the FAP binder provided herein encoded by the vector (e.g., an anti-FAP antibody or its antigen-binding fragment, a bispecific antibody containing it, or the aforementioned fusion protein). The polynucleotides contained in the vector replicate during host cell division, thereby amplifying the nucleic acids contained herein.

為了重組產生FAP結合劑，單離編碼FAP結合劑的核酸，例如如上所述，且將其插入一或多個載體中，用於在宿主細胞中進一步選殖及/或表現。此類核酸可易於使用習知程序（例如藉由使用能夠特異性結合編碼FAP結合劑的重鏈及輕鏈之基因的寡核苷酸探針）單離及定序，或由重組方法產生，或由化學合成獲得。To recombinantly generate FAP binders, nucleic acids encoding FAP binders are isolated, such as those described above, and inserted into one or more vectors for further selection and/or expression in host cells. Such nucleic acids can be readily isolated and sequenced using familiar procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of FAP binders), or generated by recombinant methods, or obtained through chemical synthesis.

將一或多種核酸導入所欲宿主細胞可由任何方法完成，包括但不限於磷酸鈣轉染、DEAE-右旋糖酐介導的轉染、陽離子脂質介導的轉染、電穿孔、轉導、感染等。非限制性例示性方法描述於例如Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press (2001)中。根據任何適合的方法，核酸可瞬時或穩定地轉染到所欲宿主細胞中。因此，用於建構表現系統的方法應為所屬技術領域中具有通常知識者已知的，例如包括但不限於顯微注射、基因槍法、電穿孔、病毒介導的轉化、電子轟擊、磷酸鈣沉澱、或其組合。 The introduction of one or more nucleic acids into a desired host cell can be accomplished by any method, including but not limited to calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, etc. Non-limiting illustrative methods are described, for example, in Sambrook et al ., Molecular Cloning, A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press (2001). Nucleic acids can be transiently or stably transfected into the desired host cell according to any suitable method. Therefore, methods used to construct phenotype systems should be known to those of ordinary skill in the art, including but not limited to microinjection, gene gun techniques, electroporation, virus-mediated transformation, electron bombardment, calcium phosphate precipitation, or combinations thereof.

任何適合表現載體表現的細胞均可用作宿主細胞。例如，宿主細胞可係原核細胞，諸如細菌細胞；或低等真核細胞，諸如酵母菌細胞；或高等真核細胞，諸如哺乳動物細胞，具體包括但不限於大腸桿菌、鏈黴菌；鼠傷寒沙門氏菌的細菌細胞；或真菌細胞，諸如酵母菌及絲狀真菌；植物細胞；衍生自果蠅S2或Sf9的昆蟲細胞；動物細胞，諸如CHO、COS、HEK293細胞、或 Bowes黑色素瘤細胞、或其組合。 Any cell suitable for expression on a vector can be used as a host cell. For example, host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells, including but not limited to Escherichia coli, Streptococcus; Salmonella typhimurium bacterial cells; or fungal cells, such as yeast and filamentous fungi; plant cells; insect cells derived from fruit fly S2 or Sf9; animal cells, such as CHO, COS, HEK293 cells, or Bowes melanoma cells, or combinations thereof.

例如，抗體可在細菌中產生，特別係當不需要糖基化及Fc效應功能時。關於細菌中抗體片段及多肽的表現，參見例如美國專利第5,648,237號、第5,789,199號、及第5,840,523號。（亦參見Charlton, K. A., In: Methods in Molecular Biology, Vol. 248, Lo, B. K. C. (ed.), Humana Press, Totowa, N.J., pp. 245-254 (2003)，其描述抗體片段在大腸桿菌中的表現）表現之後，抗體可從細菌細胞糊以可溶流份單離且可進一步純化。For example, antibodies can be generated in bacteria, especially when glycosylation and Fc effector function are not required. For the expression of antibody fragments and peptides in bacteria, see, for example, US Patents 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, K. A., In: Methods in Molecular Biology, Vol. 248, Lo, B. K. C. (ed.), Humana Press, Totowa, N.J., pp. 245-254 (2003), which describes the expression of antibody fragments in *E. coli*.) After expression, antibodies can be isolated from bacterial cell paste in soluble fractions and can be further purified.

除原核生物之外，諸如絲狀真菌或酵母菌的真核微生物亦係FAP結合劑編碼載體的適合選殖或表現宿主，包括糖基化路徑已「人源化」的真菌菌株及酵母菌菌株，導致產生具有部分或完全人類糖基化模式的FAP結合劑（例如抗FAP抗體或其抗原結合片段、雙特異性抗體、或其融合蛋白）。參見Gemgross, T. U., Nat. Biotech. 22:1409-1414 (2004)；及Li, H. et al., Nat. Biotech. 24:210-215 (2006)。 Besides prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are also suitable hosts for the colonization or expression of FAP-binding vectors, including fungal and yeast strains with "humanized" glycosylation pathways, resulting in FAP-binding agents with partial or complete human glycosylation patterns (e.g., anti-FAP antibodies or their antigen-binding fragments, bispecific antibodies, or their fusion proteins). See Gemgross, TU, Nat. Biotech. 22:1409-1414 (2004); and Li, H. et al ., Nat. Biotech. 24:210-215 (2006).

用於表現（糖基化）FAP結合劑的適合宿主細胞亦衍生自多細胞生物體（無脊椎動物及脊椎動物）。無脊椎動物細胞的實例包括植物細胞及昆蟲細胞。已識別出許多可與昆蟲細胞結合使用的桿狀病毒株，特別係用於轉染草地貪夜蛾細胞。Suitable host cells for expressing (glycosylated) FAP binders are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Many baculovirus strains that can be used in combination with insect cells have been identified, particularly for transfecting fall armyworm cells.

植物細胞培養物亦可用作宿主。參見例如美國專利第5,959,177號、第6,040,498號、第6,420,548號、第7,125,978號、及第6,417,429號（描述了用於在轉殖基因植物中產生抗體的PLANTIBODIES ™技術）。 Plant cell cultures can also be used as hosts. See, for example, U.S. Patents 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe PLATNIBODIES ™ technology for generating antibodies in transgenic plants).

脊椎動物細胞亦可用作宿主。例如，適合在懸浮液中生長的哺乳動物細胞系可能係有用的。有用的哺乳動物宿主細胞系的其他實例係由SV40 (COS-7)轉化的猴腎CV1系；人類胚胎腎系（293或293T細胞，例如如Graham, F. L. et al., J. Gen Virol.36:59-74 (1977)中所述）或Epi293細胞，如本文中所使用；小倉鼠腎細胞(baby hamster kidney cell, BHK)；小鼠塞特利氏細胞（TM4細胞，例如如Mather, J. P., Biol. Reprod. 23: 243-252 (1980)中所述）；猴腎細胞(CV1)；非洲綠猴腎細胞(VERO-76)；人類子宮頸癌細胞(HELA)；犬腎細胞(MDCK；水牛鼠肝細胞(BRL 3A)；人類肺細胞(W138)；人類肝細胞(Hep G2)；小鼠乳房腫瘤(MMT 060562)；TRI細胞（例如如Mather, J. P. et al., Annals N.Y. Acad. Sci. 383: 44-68 (1982)中所述）；MRC 5細胞；及FS4細胞。其他有用的哺乳動物宿主細胞系包括中國倉鼠卵巢(Chinese Hamster Ovary, CHO)細胞，包括DHFR-CHO細胞(Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77: 4216-4220 (1980))；及骨髓瘤細胞系，諸如Y0、NS0、及Sp2/0。有關適合抗體生產的某些哺乳動物宿主細胞系的綜述，參見例如Yazaki, P. and Wu, A. M., Methods in Molecular Biology, Vol. 248, Lo, B. K. C. (ed.), Humana Press, Totowa, N.J., pp. 255-268 (2004)。在一些實施例中，宿主細胞係真核細胞，例如中國倉鼠卵巢(CHO)細胞或淋巴細胞（例如Y0、NS0、Sp20細胞）。 Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are the monkey kidney CV1 line transformed from SV40 (COS-7); human embryonic kidney lines (293 or 293T cells, e.g., as described in Graham, FL et al ., J. Gen Virol. 36:59-74 (1977)) or Epi293 cells, as used herein; baby hamster kidney cells (BHK); and mouse Settly cells (TM4 cells, e.g., Mather, JP, Biol. Reprod. 23: 243-252). (as described in 1980); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells (e.g., as described in Mather, JP et al ., Annals NY Acad. Sci. 383: 44-68 (1982)); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (Chinese hamster ovary, CHO cells, including DHFR-CHO cells (Urlaub, G. et al ., Proc. Natl. Acad. Sci. USA 77: 4216-4220 (1980)); and myeloma cell lines, such as Y0, NSO, and Sp2/0. For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki, P. and Wu, AM, Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ, pp. 255-268 (2004). In some embodiments, the host cells are eukaryotic cells, such as Chinese hamster ovary (CHO) cells or lymphocytes (e.g., Y0, NSO, Sp20 cells).

本文所提供之FAP結合劑可由任何適合的方法純化。此類方法包括但不限於使用親和基質或疏水性交互作用層析。適合的親和配體包括ROR1 ECD及結合抗體恆定區的配體。例如，蛋白質A、蛋白質G、蛋白質A/G、或抗體親和柱可用以結合恆定區或Fc區並純化抗體或融合蛋白。疏水性交互作用層析（例如丁基或苯基柱）亦可能適合純化一些多肽。離子交換層析（例如陰離子交換層析及/或陽離子交換層析）亦可適合純化一些多肽。混合模式層析（例如反相/陰離子交換、反相/陽離子交換、親水性交互作用/陰離子交換、親水性交互作用/陽離子交換等）亦可能適合純化一些多肽。許多純化多肽的方法係所屬技術領域中已知的。The FAP binding agent described herein can be purified by any suitable method. Such methods include, but are not limited to, the use of affinity matrices or hydrophobic interaction chromatography. Suitable affinity ligands include ROR1 ECD and ligands that bind to the stationary region of the antibody. For example, protein A, protein G, protein A/G, or antibody affinity columns can be used to bind the stationary or Fc region and purify the antibody or fusion protein. Hydrophobic interaction chromatography (e.g., butyl or phenyl columns) may also be suitable for purifying some peptides. Ion exchange chromatography (e.g., anion exchange chromatography and/or cation exchange chromatography) may also be suitable for purifying some peptides. Mixed-mode chromatography (e.g., reversed-phase/anion exchange, reversed-phase/cation exchange, hydrophilic interaction/anion exchange, hydrophilic interaction/cation exchange, etc.) may also be suitable for purifying some peptides. Many methods for purifying peptides are known in the relevant technical field.

在一些實施例中，FAP結合劑在無細胞系統中產生。非限制性例示性無細胞系統描述於例如Sitaraman et al., Methods Mol. Biol. 498: 229-44 (2009)；Spirin, Trends Biotechnol. 22: 538-45 (2004)；Endo et al., Biotechnol. Adv. 21: 695-713 (2003)。 使用的系統 In some embodiments, the FAP binder is generated in a cell-free system. Non-limiting illustrative cell-free systems are described, for example, in Sitaraman et al ., Methods Mol. Biol. 498: 229-44 (2009); Spirin, Trends Biotechnol. 22: 538-45 (2004); Endo et al ., Biotechnol. Adv. 21: 695-713 (2003). Systems used .

本文提供使用本文所述之FAP結合劑的系統。本文所提供之系統包含組分，其中組分包含：本文所提供之FAP結合劑中之任一或多者，包括抗FAP抗體或其抗原結合片段、包含其之雙特異性、本文所述之融合蛋白、其變體、其衍生化形式、編碼其之核酸、及/或包含此類核酸的載體。This document provides a system for using the FAP binders described herein. The system provided herein comprises components, wherein the components include: any or more of the FAP binders provided herein, including anti-FAP antibodies or antigen-binding fragments thereof, bispecificity thereof, the fusion proteins described herein, variants thereof, derivatized forms thereof, nucleic acids encoding thereof, and/or vectors comprising such nucleic acids.

在一些實施例中，本文所提供之系統進一步包含自對象獲得的樣本。樣本係生物樣本。在一些實施例中，樣本係獲自有需要用本文所提供之FAP結合劑治療之對象。在一些實施例中，樣本係腫瘤樣本。In some embodiments, the system provided herein further includes samples obtained from the subject. The samples are biological samples. In some embodiments, the samples are obtained from subjects who require treatment with the FAP conjugate provided herein. In some embodiments, the samples are tumor samples.

在一些實施例中，本文所提供之系統進一步包含一或多種系統溶液，該一或多種系統溶液包含組成物、醫藥組成物、緩衝液、包括偵測及/或擴增試劑的試劑、或其任何組合。本文進一步描述此類組成物、醫藥組成物、緩衝液、及試劑。在一些實施例中，本文所提供之系統進一步包含一或多種支撐介質、容器、及其他製品、或其任何組合。本文進一步描述此類支撐介質、容器、及其他製品。In some embodiments, the system provided herein further comprises one or more system solutions comprising components, pharmaceutical components, buffers, reagents including detection and/or amplification reagents, or any combination thereof. Such components, pharmaceutical components, buffers, and reagents are further described herein. In some embodiments, the system provided herein further comprises one or more support media, containers, and other articles, or any combination thereof. Such support media, containers, and other articles are further described herein.

在一些實施例中，本文所述之系統組分係各自包含在組成物中，或組分之任何組合係包含在單一組成物中。在一些實施例中，（例如組成物中之）本文所述之系統組分係各自包含在容器中，或組分之任何組合係包含在單一容器中。在一些實施例中，系統包含套組。在一些實施例中，包含套組的系統稱為套組。在一些實施例中，系統包含裝置。在一些實施例中，包含裝置的系統稱為裝置。 組成物 In some embodiments, the system components described herein are each contained in an assembly, or any combination of components is contained in a single assembly. In some embodiments, (e.g., within an assembly) the system components described herein are each contained in a container, or any combination of components is contained in a single container. In some embodiments, the system includes a set. In some embodiments, a system including a set is called a set. In some embodiments, the system includes a device. In some embodiments, a system including a device is called a device. Assembly

本文提供可用於偵測FAP、診斷FAP相關疾病或病症、介導以FAP及/或FAP表現性細胞的表現為特徵的患病微環境中的活性、治療FAP相關疾病或病症、或其任何組合的組成物。本文所提供之組成物包含：本文所提供之FAP結合劑中之任一或多者，包括抗FAP抗體、其抗原結合片段、其變體、其衍生化形式、及針對其之結合多肽、編碼其之核酸、及/或包含此類核酸的載體。可選地，組成物包含本文所述之緩衝液、試劑、載劑、賦形劑、及穩定劑。This document provides compositions for detecting FAP, diagnosing FAP-related diseases or conditions, mediating activity in diseased microenvironments characterized by the expression of FAP and/or FAP-expressing cells, treating FAP-related diseases or conditions, or any combination thereof. The compositions provided herein include any or more of the FAP conjugates provided herein, including anti-FAP antibodies, their antigen-binding fragments, their variants, their derivatized forms, and binding peptides targeting them, nucleic acids encoding them, and/or vectors containing such nucleic acids. Optionally, the compositions include buffers, reagents, carriers, adjuvants, and stabilizers described herein.

在一些實施例中，組成物係包含FAP結合劑的細胞培養基。在一些實施例中，宿主細胞培養液包含FAP結合劑。在一些實施例中，組成物係包含FAP結合劑的偵測劑。在一些實施例中，組成物係包含FAP結合劑的醫藥組成物。 醫藥組成物 In some embodiments, the formulation is a cell culture medium containing an FAP conjugate. In some embodiments, the host cell culture medium contains an FAP conjugate. In some embodiments, the formulation is a detector containing an FAP conjugate. In some embodiments, the formulation is a pharmaceutical composition containing an FAP conjugate. Pharmaceutical Composition

本揭露係關於一種醫藥組成物，其包括上文所描述之抗FAP抗體或其抗原結合片段、及醫藥上可接受之載劑。This disclosure relates to a pharmaceutical composition comprising the anti-FAP antibody or antigen-binding fragment thereof described above, and a pharmaceutically acceptable delivery vehicle.

本揭露係關於一種醫藥組成物，其包括上文所描述之融合蛋白、及醫藥上可接受之載劑。醫藥上可接受之載劑包括但不限於緩衝液、賦形劑、穩定劑、或防腐劑。此類載劑包括（但不限於）鹽水、緩衝液、葡萄糖、水、甘油、乙醇、及其組合。所採用的載劑、賦形劑、或穩定劑通常對於在所使用的劑量及濃度下暴露於其的細胞或哺乳動物係無毒的。通常，載劑係pH緩衝水溶液。載劑之實例包括緩衝液，諸如磷酸鹽、檸檬酸鹽、及其他有機酸；抗氧化劑，包括抗壞血酸；低分子量（例如少於約10個胺基酸殘基）多肽；蛋白質，諸如血清白蛋白、明膠、或免疫球蛋白；親水性聚合物，諸如聚乙烯吡咯啶酮；胺基酸，諸如甘胺酸、麩醯胺酸、天冬醯胺酸、精胺酸、或離胺酸；單醣、雙醣、及其他碳水化合物，包括葡萄糖、甘露糖、或糊精；螯合劑，諸如EDTA；糖醇，諸如甘露醇或山梨醇；成鹽相對離子，諸如鈉；及/或非離子界面活性劑，諸如TWEEN ™、聚乙二醇(PEG)、及PLURONICS ™。 This disclosure relates to a pharmaceutical composition comprising the fusion protein described above and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include, but are not limited to, buffers, adsorbents, stabilizers, or preservatives. Such carriers include, but are not limited to, brine, buffers, glucose, water, glycerol, ethanol, and combinations thereof. The carrier, adsorbent, or stabilizer used is generally non-toxic to cells or mammals exposed to it at the dosage and concentration used. Typically, the carrier is a pH-buffered aqueous solution. Examples of carriers include buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (e.g., fewer than about 10 amino acid residues) peptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamic acid, aspartic acid, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming ions such as sodium; and/or nonionic surfactants such as TWEEN ™. Polyethylene glycol (PEG) and PLURONICS ™ .

載劑亦可指稀釋劑、佐劑（例如弗氏佐劑（完全或不完全））、賦形劑、或與治療劑一起投予的媒劑。此類載劑可係無菌液體，諸如水及油，包括石油、動物、植物、或合成來源之無菌液體，諸如花生油、大豆油、礦物油、芝麻油、及類似者。當組成物（例如醫藥組成物）經由靜脈內投予時，水係例示性載劑。鹽水溶液及右旋糖水性溶液及甘油溶液亦可用作液體載劑，特別係用於注射溶液。適合的賦形劑（例如醫藥賦形劑）包括澱粉、葡萄糖、乳糖、蔗糖、明膠、麥芽、大米、麵粉、白土粉、矽膠、硬脂酸鈉、甘油單硬脂酸酯、滑石、氯化鈉、乾燥之脫脂牛奶、甘油、丙烯、乙二醇、水、乙醇、及類似物。若為所欲，組成物亦可含有少量的潤濕劑或乳化劑，或pH緩衝劑。A carrier can also refer to a diluent, adjuvant (e.g., Freund's adjuvant (complete or incomplete)), excipient, or a medium administered with a treatment. Such carriers can be sterile liquids, such as water and oils, including sterile liquids of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and similar substances. Water is an exemplary carrier when a composition (e.g., a pharmaceutical composition) is administered intravenously. Saline solutions, dextran aqueous solutions, and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable excipients (e.g., pharmaceutical excipients) include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, kaolin, silicone, sodium stearate, glyceryl monostearate, talc, sodium chloride, dried skim milk, glycerin, propylene, ethylene glycol, water, ethanol, and similar substances. The composition may also contain small amounts of wetting agents or emulsifiers, or pH buffers, if desired.

通常，此等物質可配製在無毒、惰性、及醫藥上可接受之水性載劑介質中，其中pH通常係約5至8，較佳地約6至8，儘管pH可根據所配製物質的性質及待治療的疾病病況而改變。Typically, these substances are formulated in non-toxic, inert, and pharmaceutically acceptable aqueous carrier media, with a pH usually of about 5 to 8, preferably about 6 to 8, although the pH may vary depending on the nature of the substance being formulated and the condition of the disease being treated.

組成物可採取溶液、懸浮液、乳液、緩釋製劑、及類似者的形式。投予包含抗體的組成物的適合途徑係所屬技術領域中熟知的。儘管可使用多於一種途徑來投予FAP結合劑（例如抗FAP抗體、FAP抗原結合部份、包含FAP抗原結合部份之雙特異性抗體、或其融合蛋白），但特定途徑可提供比另一途徑更直接及更有效的反應。取決於情況，將包含FAP結合劑（例如抗FAP抗體、FAP抗原結合部份、包含FAP抗原結合部份之雙特異性抗體、或其融合蛋白）（諸如人類FAP結合劑）之組成物施用或滴注至體腔中及/或引入到循環中。The composition may be in the form of solution, suspension, emulsion, sustained-release formulation, and similar forms. Suitable routes of administration for compositions containing antibodies are well known in the art. Although more than one route may be used to administer FAP conjugates (e.g., anti-FAP antibodies, FAP antigen-binding moieties, bispecific antibodies containing FAP antigen-binding moieties, or fusion proteins thereof), a particular route may provide a more direct and effective response than another. Depending on the circumstances, compositions containing FAP conjugates (e.g., anti-FAP antibodies, FAP antigen-binding moieties, bispecific antibodies containing FAP antigen-binding moieties, or fusion proteins thereof) (such as human FAP conjugates) may be administered or infused into body cavities and/or introduced into circulation.

例如，可能所欲的係透過靜脈內、皮下、腹膜內、腦內（實質內）、腦室內、肌內、動脈內、門靜脈內、病灶內、髓內、鞘內、心室內、經皮、皮下、藉由緩釋系統、或藉由植入裝置的注射來遞送包含FAP結合劑的組成物。若為所欲，FAP結合劑經由供給所關注之區的動脈內或靜脈內投予（例如經由肝動脈以遞送至肝臟）來局部投予。在其他態樣中，FAP結合劑在腫瘤切除或其他外科程序期間直接投予至暴露的組織。For example, the desired delivery method may be intravenous, subcutaneous, intraperitoneal, intracranial (intra-parenchymal), intraventricular, intramuscular, intraarterial, intraportal, intralesional, intramedullary, intrasheath, intraventricular, percutaneous, subcutaneous, via a sustained-release system, or via injection through an implanted device. If desired, the FAP conjugate may be administered locally via intra-arterial or intravenous administration to the area of concern (e.g., via the hepatic artery to the liver). In other cases, the FAP conjugate may be administered directly to exposed tissue during tumor resection or other surgical procedures.

如本文所述，所配製的醫藥組成物可由習知途徑投予，包括（但不限於）腫瘤內投予、腹膜內投予、靜脈內投予、或局部投予。醫藥製劑應與投予方式相匹配。本申請案的醫藥組成物可製備成注射劑形式，例如，醫藥組成物係以生理鹽水或含有葡萄糖及其他佐劑的水溶液按照習知方法製備。As described herein, the formulated pharmaceutical composition may be administered via known routes, including (but not limited to) intratumoral administration, intraperitoneal administration, intravenous administration, or local administration. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical composition of this application may be prepared in injectable form, for example, by means of physiological saline or an aqueous solution containing glucose and other adjuvants prepared according to known methods.

醫藥組成物（諸如注射劑及溶液）應在無菌條件下製造。Pharmaceutical components (such as injections and solutions) should be manufactured under sterile conditions.

本揭露之醫藥組成物含有安全且有效量（諸如0.001至99 wt%、0.01至95 wt%、或0.1至90 wt%）的所提供之單域抗體或融合蛋白及醫藥上可接受之載劑或賦形劑。The pharmaceutical composition disclosed herein contains a safe and effective amount (e.g., 0.001 to 99 wt%, 0.01 to 95 wt%, or 0.1 to 90 wt%) of the provided single-domain antibody or fusion protein and a pharmaceutically acceptable carrier or adjuvant.

在一些實施例中，活性成分的劑量係治療有效量，諸如每天約10 µg/kg體重至約100 mg/kg體重。此外，FAP結合劑（例如抗FAP抗體或其抗原結合片段、包含其的雙特異性抗體、或上述融合蛋白）亦可與其他治療劑一起使用。因此，活性成分的劑量可指單獨或與其他治療劑組合的FAP結合劑（例如抗FAP抗體或其抗原結合片段、包含其的雙特異性抗體、或上述融合蛋白）的量。與本文所提供之系統、組成物、及/或醫藥組成物相容的額外治療劑在本文的組合治療部分中進一步描述。 偵測及診斷 In some embodiments, the dosage of the active ingredient is a therapeutically effective amount, such as approximately 10 µg/kg body weight to approximately 100 mg/kg body weight daily. Furthermore, FAP conjugates (e.g., anti-FAP antibodies or their antigen-binding fragments, bispecific antibodies containing them, or the aforementioned fusion proteins) may also be used in combination with other therapeutic agents. Therefore, the dosage of the active ingredient may refer to the amount of an FAP conjugate (e.g., anti-FAP antibodies or their antigen-binding fragments, bispecific antibodies containing them, or the aforementioned fusion proteins), alone or in combination with other therapeutic agents. Additional therapeutic agents compatible with the systems, compositions, and/or pharmaceutical compositions provided herein are further described in the Combination Therapy section of this document. Detection and Diagnosis

本揭露提供使用本文所提供之FAP結合劑（例如抗FAP抗體或其抗原結合片段、雙特異性抗體、或其融合蛋白）、經單離多核苷酸、構築體、及/或醫藥組成物偵測及診斷對象中的疾病或病症（例如腫瘤疾病）的方法。在一些實施例中，用於偵測及診斷的方法可用以判定本文所述之抗體或多肽是否係針對對象的適當治療。This disclosure provides methods for detecting and diagnosing diseases or conditions (e.g., oncological diseases) in subjects using FAP conjugates (e.g., anti-FAP antibodies or their antigen-binding fragments, bispecific antibodies, or their fusion proteins) described herein, via isolated polynucleotides, constructs, and/or pharmaceutical compositions. In some embodiments, the methods used for detection and diagnosis can be used to determine whether the antibodies or peptides described herein are appropriate treatments for the subject.

可分析來自對象（例如懷疑患有或已知患有具有FAP表現之腫瘤疾病的個體、或懷疑患有或已知患有另一疾病或病況的個體）之樣本（例如測試生物樣本）的FAP存在、不存在、表現、及/或水平。例如，可藉由偵測本文所提供之FAP結合劑（例如抗FAP抗體或其抗原結合片段、雙特異性抗體、或其融合蛋白）與樣本中的物質（例如蛋白質）結合的存在或不存在來收集及分析此類樣本。在一些實例中，方法進一步包括將偵測到的結合量與對照樣本的結合量比較，或將偵測到的FAP水平與FAP的對照水平比較。在一些情況下，方法指示FAP相關疾病或病況（諸如本文所述之疾病或病況）的存在、不存在、或嚴重性。This method can analyze the presence, absence, manifestation, and/or level of FAP in samples (e.g., test biological samples) from individuals (e.g., individuals suspected of having or known to have a tumor with FAP manifestations, or individuals suspected of having or known to have another disease or condition). For example, such samples can be collected and analyzed by detecting the presence or absence of binding between a FAP binder (e.g., an anti-FAP antibody or its antigen-binding fragment, a bispecific antibody, or its fusion protein) provided herein and a substance (e.g., a protein) in the sample. In some instances, the method further includes comparing the detected amount of binding to the amount of binding in a control sample, or comparing the detected FAP level to a control level of FAP. In some cases, the method indicates the presence, absence, or severity of FAP-related diseases or conditions (such as those described herein).

此分析可在使用本文所提供之FAP結合劑（例如抗FAP抗體或其抗原結合片段、雙特異性抗體、或其融合蛋白）、經單離多核苷酸、構築體、及/或醫藥組成物起始治療前執行，或可作為監測癌症治療進展的一部分進行。在一些實施例中，提供治療方法，其藉由執行偵測檢定且例如基於診斷檢定之結果起始、改變、或中止對象之治療來進行。此類診斷分析可使用任何樣本執行，包括但不限於組織、從此類組織單離的細胞、及類似者。在一些情況下，方法係對液體樣本執行，例如血液、血漿、血清、全血、唾液、尿液、或精液。組織樣本包括例如福馬林固定或冷凍的組織切片。This analysis can be performed before initiating treatment with the FAP binding agents provided herein (e.g., anti-FAP antibodies or their antigen-binding fragments, bispecific antibodies, or fusion proteins thereof), via isolated polynucleotides, constructs, and/or pharmaceutical compositions, or as part of monitoring cancer treatment progress. In some embodiments, treatment methods are provided that initiate, modify, or discontinue treatment of a subject by performing a detection assay, for example, based on the results of a diagnostic assay. Such diagnostic analyses can be performed using any sample, including but not limited to tissues, cells isolated from such tissues, and similar samples. In some cases, the method is performed on liquid samples, such as blood, plasma, serum, whole blood, saliva, urine, or semen. Tissue samples include, for example, formalin-fixed or frozen tissue sections.

可採用任何適合的方法來偵測及分析FAP。所屬技術領域中已知的各種診斷檢定技術可適合用於此類目的，諸如競爭性結合檢定、直接或間接夾心檢定、及在異相或均相中進行的免疫沉澱檢定。用於執行上述各種免疫檢定的一般技術係所屬技術領域中具有通常知識者已知的。FAP can be detected and analyzed by any suitable method. Various diagnostic assays known in the art are suitable for this purpose, such as competitive binding assays, direct or indirect sandwich assays, and immunoprecipitation assays performed in heterogeneous or homogeneous phases. General techniques for performing the above-mentioned immunoassays are known to those skilled in the art.

提供用於偵測及診斷方法的FAP結合劑（例如抗FAP抗體或其抗原結合片段、雙特異性抗體、或其融合蛋白）可用可偵測部份標記。可偵測部份直接或間接產生可偵測信號。例如，可偵測部分可係本文所述者中之任一者，例如放射性同位素（諸如3H、14C、32P、35S、或125I）、螢光或化學發光化合物（諸如螢光異硫氰酸鹽(fluorescein isothiocyanate, FITC)、Texas紅、花青、光青、玫瑰紅、或螢光素）、或酶（諸如鹼性磷酸酶、β-半乳糖苷酶、或辣根過氧化酶）。FAP-binding agents (e.g., anti-FAP antibodies or their antigen-binding fragments, bispecific antibodies, or their fusion proteins) used for detection and diagnostic methods may be labeled with a detectable moiety. The detectable moiety generates a detectable signal directly or indirectly. For example, the detectable moiety may be any of those described herein, such as a radioactive isotope (e.g., 3H, 14C, 32P, 35S, or 125I), a fluorescent or chemiluminescent compound (e.g., fluorescein isothiocyanate (FITC), Texas red, anthocyanin, cyanine, rose red, or fluorescein), or an enzyme (e.g., alkaline phosphatase, β-galactosidase, or horseradish peroxidase).

偵測可藉由在適合向FAP提供FAP結合劑（例如抗FAP抗體或其抗原結合片段、雙特異性抗體、或其融合蛋白）的條件下接觸樣本並評估樣本中FAP的存在（例如水平）來完成。樣本中的FAP水平與參考樣本的水平相比可指示具有FAP活性的腫瘤或腫瘤相關組織的存在。參考樣本可係在較早時間點取自對象的樣本或來自另一個體的樣本。 套組 Detection can be performed by accessing a sample under conditions suitable for supplying FAP binders (e.g., anti-FAP antibodies or their antigen-binding fragments, bispecific antibodies, or their fusion proteins) and assessing the presence (e.g., level) of FAP in the sample. The FAP level in the sample, compared to the level in a reference sample, indicates the presence of a tumor or tumor-related tissue with FAP activity. The reference sample can be a sample taken from the subject at an earlier time or a sample from another individual. Kit

本揭露提供一種套組，其含有本文所提供之FAP結合劑（例如抗FAP抗體或其抗原結合片段、雙特異性抗體、或其融合蛋白）、經單離多核苷酸、構築體、及/或醫藥組成物。若為所欲，此類套組可進一步包括各種習知醫藥套組組分中的一或多者，諸如例如具有一或多種醫藥上可接受之載劑的容器、額外的容器等，這對於所屬技術領域中具有通常知識者而言將係顯而易見的。套組中亦可包括指示待投予組分的量、投予指南、及/或混合組分指南的說明書，作為插頁或標籤。在一些實施例中，本文所提供之套組進一步包括用於使用本文所提供之經單離多核苷酸、構築體、及/或醫藥組成物用於本文所提供之方法中的說明書。儘管含有說明書之電子儲存媒體（例如磁碟或光碟）亦為可接受的，但使用說明通常係書面說明書。This disclosure provides a kit containing the FAP conjugates (e.g., anti-FAP antibodies or their antigen-binding fragments, bispecific antibodies, or their fusion proteins) provided herein, as well as isolated polynucleotides, constructs, and/or pharmaceutical components. If desired, such kits may further include one or more of various known pharmaceutical kit components, such as containers having one or more pharmaceutically acceptable carriers, additional containers, etc., as will be apparent to those skilled in the art. The kit may also include instructions indicating the amount of components to be administered, administration guidelines, and/or instructions for mixing components, as inserts or labels. In some embodiments, the kits provided herein further include instructions for use of the isolated polynucleotides, structures, and/or pharmaceutical compositions provided herein in the methods provided herein. Although electronic storage media containing instructions (such as magnetic disks or optical disks) are also acceptable, the instructions are generally written instructions.

在一些實施例中，套組包含用於本文所述方法之適合包裝或容器中之本文所提供之FAP結合劑（例如抗FAP抗體或其抗原結合片段、雙特異性抗體、或其融合蛋白）、經單離多核苷酸、構築體、及/或醫藥組成物。適合的包裝及容器係所屬技術領域中已知的，且包括例如小瓶、容器、安瓿、瓶子、注射器（例如載藥注射器）、廣口瓶、可撓性包裝、及類似者。製品可進一步經滅菌及/或密封。各組分（若存在多於一種組分）可包裝於單獨容器中，或在交叉反應性及存放期准許之情況下可將一些組分合併於一個容器中。套組可呈單位劑型、批量包裝（例如多劑量包裝）、或次單位劑量。套組亦可包括多個單位劑量的化合物及使用說明，且可以足以在藥局（例如醫院藥局及調製藥局）中儲存及使用之數量包裝。In some embodiments, the kit comprises the FAP conjugate (e.g., an anti-FAP antibody or its antigen-binding fragment, a bispecific antibody, or a fusion protein thereof), isolated polynucleotide, construct, and/or pharmaceutical composition provided herein, in a suitable package or container for use with the methods described herein. Suitable packages and containers are known in the art and include, for example, vials, containers, ampoules, bottles, syringes (e.g., drug-loaded syringes), wide-mouth bottles, flexible packaging, and the like. The product may be further sterilized and/or sealed. Components (if more than one component is present) may be packaged in individual containers, or some components may be combined in one container where cross-reactivity and shelf life permit. The kit may be in unit dosage form, batch packaging (e.g., multi-dose packaging), or subunit dosage form. The kit may also include multiple unit doses of the compound and instructions for use, and may be packaged in quantities sufficient for storage and use in pharmacies (such as hospital pharmacies and dispensing pharmacies).

在一些實施例中，套組包含一或多個醫藥包，該等醫藥包包含一或多個容器（例如小瓶、安瓿、載藥注射器），該一或多個容器含有本文所提供之FAP結合劑（例如抗FAP抗體或其抗原結合片段、雙特異性抗體、或其融合蛋白）、經單離多核苷酸、構築體、及/或醫藥組成物。在一些情況下，套組含有本文所述之醫藥組成物。在一些實施例中，套組包含冷凍乾燥形式的本文所提供之FAP結合劑（例如抗FAP抗體或其抗原結合片段、雙特異性抗體、或其融合蛋白）、經單離多核苷酸、構築體、及/或醫藥組成物。與此類（多種）容器相關聯的亦可係呈政府機構規範藥品或生物產品之製造、使用、或銷售之規定形式的標示，該標示反映機構核准用於人類投予之製造、使用、或銷售。 使用方法 In some embodiments, the kit comprises one or more medical packets containing one or more containers (e.g., vials, ampoules, drug-loaded syringes) containing the FAP conjugate described herein (e.g., anti-FAP antibody or its antigen-binding fragment, bispecific antibody, or fusion protein thereof), via isolated polynucleotides, constructs, and/or pharmaceutical compositions. In some cases, the kit contains the pharmaceutical compositions described herein. In some embodiments, the kit comprises the FAP conjugate described herein (e.g., anti-FAP antibody or its antigen-binding fragment, bispecific antibody, or fusion protein thereof) in a freeze-dried form, via isolated polynucleotides, constructs, and/or pharmaceutical compositions. Related to this type (multiple) of containers may also be labels in the form prescribed by government agencies for the manufacture, use, or sale of pharmaceuticals or biological products, reflecting the agency's approval for manufacture, use, or sale for human consumption. Instructions for Use

本揭露提供上述FAP結合劑（例如抗FAP抗體或其抗原結合片段、雙特異性抗體、或其融合蛋白）或醫藥組成物於製造用於預防、診斷、或治療疾病、病症、或病況之治療劑的用途。因此，本文提供一種治療疾病的方法，該方法包含向有需要之對象投予有效量或劑量的FAP結合劑（例如抗FAP抗體、FAP抗原結合部份、包含FAP抗原結合部份之雙特異性抗體、或其融合蛋白），該FAP結合劑包括其修飾劑（例如接合的抗FAP抗體）、組成物、或包含其之醫藥組成物。This disclosure provides the use of the aforementioned FAP conjugates (e.g., anti-FAP antibodies or their antigen-binding fragments, bispecific antibodies, or fusion proteins thereof) or pharmaceutical compositions in the manufacture of therapeutics for the prevention, diagnosis, or treatment of diseases, conditions, or illnesses. Therefore, this document provides a method for treating a disease comprising administering to a subject in need an effective amount or dose of an FAP conjugate (e.g., an anti-FAP antibody, an FAP antigen-binding moiety, a bispecific antibody comprising an FAP antigen-binding moiety, or a fusion protein thereof), the FAP conjugate including its modifiers (e.g., conjugated anti-FAP antibodies), compositions, or pharmaceutical compositions comprising it.

治療方法亦可包含偵測獲自接受治療之對象之樣本中的FAP，及/或診斷以表現FAP之細胞為特徵或與表現FAP之細胞相關之疾病或患病微環境的方法。偵測及/或診斷的方法可藉由使用本文所提供之此類方法來執行，然而，所屬技術領域中具有通常知識者將理解，可利用任何適合的方法來偵測樣本中的FAP或診斷以表現FAP之細胞為特徵或與表現FAP之細胞相關的疾病。Treatment methods may also include detecting FAP in samples obtained from the treated subject, and/or diagnosing diseases or disease microenvironments characterized by or associated with cells expressing FAP. Detection and/or diagnosis methods may be performed using the methods described herein; however, those skilled in the art will understand that any suitable method may be used to detect FAP in a sample or diagnose diseases characterized by or associated with cells expressing FAP.

對於本文所提供之FAP結合劑可設想多種作用方式。例如，作用方式係將其FAP結合劑與抗體藥物接合物(ADC)形式的藥物接合。另一作用方式係FAP結合劑誘導SLO形成的能力。第三作用方式在於FAP結合劑誘導TLS形成的能力。額外作用方式係FAP結合劑活化CAF的能力。另一作用方式在於FAP結合劑誘導腫瘤細胞死亡的能力。本文進一步描述作用方式。Multiple mechanisms of action are conceivable for the FAP conjugates presented herein. For example, one mechanism is the conjugation of the FAP conjugate to a drug in the form of an antibody-drug conjugate (ADC). Another mechanism is the ability of the FAP conjugate to induce SLO formation. A third mechanism is the ability of the FAP conjugate to induce TLS formation. An additional mechanism is the ability of the FAP conjugate to activate CAF. Another mechanism is the ability of the FAP conjugate to induce tumor cell death. These mechanisms of action are further described herein.

在一些實施例中，FAP結合劑的有效量或劑量係指本文所述之FAP結合劑的量，或包含該FAP結合劑的組成物或醫藥組成物的量，其將引發研究者、醫師、或其他臨床醫師正在尋求的組織、系統、動物、哺乳動物、或人類的生物或醫學反應或對組織、系統、動物、哺乳動物、或人類的期望治療效果。FAP結合劑的有效量可根據諸如個體的疾病狀態、年齡、性別、及體重、及FAP+分子在個體中引發所欲反應的能力的因子而變化。有效量亦係其中治療有益效果超過FAP結合劑的任何毒性或有害效果的量。此類益處包括改善癌症的徵象或症狀。所屬技術領域中具有通常知識者可藉由使用已知技術並藉由觀察在類似情況下獲得的結果而容易地判定有效量。本文所述之FAP結合劑之有效量可以單次劑量或多次劑量投予。在判定患者的有效量時，主治醫師會考慮數個因子，包括但不限於：患者的體型（例如體重或質量）、體表面積、年齡、及一般健康狀況；涉及的具體疾病或病症；疾病或病症的程度、受累程度、或嚴重性；個體患者的反應；投予的特定化合物；投予方式；投予製劑的生體可用率特性；選擇的劑量方案；伴隨藥物的使用；及醫師所知的其他相關情況。在一些實施例中，對象係哺乳動物，包括人類及非人類動物，諸如人類、小鼠、及食蟹獼猴。In some embodiments, the effective amount or dosage of an FAP conjugate refers to the amount of the FAP conjugate described herein, or the amount of a component or pharmaceutical composition containing the FAP conjugate, that will elicit a biological or medical response or desired therapeutic effect in a tissue, system, animal, mammal, or human that an investigator, physician, or other clinician is seeking. The effective amount of an FAP conjugate can vary based on factors such as an individual's disease state, age, sex, and weight, and the ability of FAP+ molecules to elicit the desired response in an individual. An effective amount is also the amount in which a therapeutically beneficial effect outweighs any toxic or adverse effects of the FAP conjugate. Such benefits include improvement of signs or symptoms of cancer. Those with ordinary knowledge in the relevant technical field can easily determine the effective dose by using known techniques and by observing results obtained under similar conditions. The effective dose of the FAP conjugates described herein can be administered as a single dose or multiple doses. When determining the effective dose for a patient, the attending physician will consider several factors, including but not limited to: the patient's body type (e.g., weight or mass), body surface area, age, and general health condition; the specific disease or condition involved; the degree, extent, or severity of the disease or condition; the individual patient's response; the specific compound administered; the method of administration; the bioavailability characteristics of the administered formulation; the chosen dosage regimen; the use of concomitant medications; and other relevant information known to the physician. In some implementations, the subjects are mammals, including humans and non-human animals such as humans, mice, and cynomolgus monkeys.

在一些實施例中，本文提供一種用於治療患有癌症之對象的方法，其包括向該對象投予治療有效量的本文所提供之FAP結合劑（例如抗FAP抗體或其抗原結合片段、雙特異性抗體、或其融合蛋白）或醫藥組成物。In some embodiments, this document provides a method for treating a subject with cancer, which includes administering to the subject a therapeutically effective amount of the FAP conjugate provided herein (e.g., an anti-FAP antibody or its antigen-binding fragment, a bispecific antibody, or its fusion protein) or a pharmaceutical composition thereof.

在一些實施例中，「治療有效量」的本揭露提供之FAP結合劑（例如抗FAP抗體或其抗原結合片段、雙特異性抗體、或其融合蛋白）或醫藥組成物使得疾病症狀之嚴重性降低及疾病、病症、或病況之無症狀期的頻率及持續時間增加，或預防因為疾病或痛苦引起的損傷或殘疾。In some embodiments, the FAP conjugates (e.g., anti-FAP antibodies or their antigen-binding fragments, bispecific antibodies, or their fusion proteins) or pharmaceutical compositions provided in this disclosure, in the "therapeutic effective amount," reduce the severity of disease symptoms and increase the frequency and duration of asymptomatic periods of disease, condition, or illness, or prevent injury or disability caused by disease or suffering.

例如，對於FAP相關腫瘤（包括例如黑素瘤、淋巴瘤、膀胱癌、非小細胞肺癌、頭頸癌、及結腸癌）的治療，相對於未經治療的對象，在一些實施例中，「治療有效量」抑制細胞生長或腫瘤生長至少約10%、至少約20%、至少約30%、至少約40%、至少約50%、至少約60%、至少約70%、或至少約80%。For example, in the treatment of FAP-related tumors (including, for example, melanoma, lymphoma, bladder cancer, non-small cell lung cancer, head and neck cancer, and colon cancer), in some embodiments, the "therapeutic effective dose" inhibits cell growth or tumor growth by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, or at least about 80%, relative to untreated subjects.

抑制腫瘤生長的能力可在預測針對人類腫瘤的功效的動物模型系統中進行評估，或藉由偵測抑制細胞生長的能力來評估。此類抑制可藉由所屬技術領域中具有通常知識者熟知的檢定在體外判定。治療有效量的FAP結合劑（例如抗FAP抗體或其抗原結合片段、雙特異性抗體、或其融合蛋白）及醫藥組成物通常能夠減小腫瘤大小，或以其他方式緩解對象之症狀。所屬技術領域中具有通常知識者可根據實際情況（例如對象之腫瘤大小、對象症狀之嚴重性、及所選特定組成物或投予途徑）選擇適合的治療有效劑量。治療處方（例如劑量決定等）可由醫師通常考慮各因子判定，該等因子包括但不限於所治療之疾病、患者之狀態、遞送部位、投予途徑、及其他因子。預防有效量(prophylactically effective amount)係指在所需劑量及一段時間內有效達到所欲預防效果的量。通常但不必然，由於預防劑量係在疾病發作之前或在疾病的早期階段向對象投予的，因此「預防有效量」通常低於「治療有效量」。The ability to inhibit tumor growth can be assessed in animal model systems that predict efficacy against human tumors, or by detecting the ability to inhibit cell growth. Such inhibition can be determined in vitro using tests well-known to those skilled in the art. Therapeutic doses of FAP conjugates (e.g., anti-FAP antibodies or their antigen-binding fragments, bispecific antibodies, or their fusion proteins) and pharmaceutical compositions typically reduce tumor size or otherwise relieve symptoms. Those skilled in the art can select appropriate therapeutic doses based on the specific circumstances (e.g., tumor size, severity of symptoms, and the chosen composition or route of administration). Treatment prescriptions (such as dosage determination) are typically determined by physicians considering various factors, including but not limited to the disease being treated, the patient's condition, the site of administration, the route of delivery, and other factors. The prophylactically effective amount refers to the dose that effectively achieves the desired preventative effect within the required timeframe. Usually, but not always, because the preventative dose is administered before the onset of disease or in its early stages, the "prophylactically effective amount" is usually lower than the "therapeutic effective amount."

對於治療應用，FAP結合劑可以醫藥上可接受之劑型投予患者或對象，例如投予人類或非人類對象。例如，投予可作為推注或藉由在一段時間內連續輸注、經由肌肉、腹膜內、腦脊髓內、皮下、關節內、滑膜內、或鞘內在靜脈內發生。根據本揭露的FAP結合劑及其醫藥組成物特別適合經由腫瘤內、腫瘤周圍、病灶內、或病灶周圍途徑進行投予，以發揮局部以及全身性治療效果。For therapeutic applications, FAP conjugates can be administered to patients or subjects in pharmaceutically acceptable dosage forms, such as human or non-human subjects. For example, administration can be by bolus injection or by continuous infusion over a period of time, via intramuscular, intraperitoneal, intraspinal, subcutaneous, intra-articular, intrasynovial, or intrathecal intravenous routes. The FAP conjugates and pharmaceutical compositions thereof according to this disclosure are particularly suitable for administration via intratumoral, peritumoral, intralesional, or perilesional routes to achieve both local and systemic therapeutic effects.

例示性投予途徑包括腸胃外（例如肌內、靜脈內、動脈內、腹膜內、或皮下）。此外，抗體、片段、接合物、及醫藥組成物可經由脈衝輸注投予，例如，減少抗體、片段、或接合物的劑量。在一些實施例中，經由注射、靜脈內、或皮下注射給予劑量，部分取決於投予係短暫的抑或慢性的。投予的量可取決於多種因子，諸如臨床症狀、患者或對象的體重、及是否投予其他藥物。技術人員將認識到，投予途徑將取決於待治療的病症或病況而變化。Examples of routes of administration include parenteral (e.g., intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous). In addition, antibodies, fragments, conjugates, and pharmaceutical compositions can be administered via intravenous infusion, for example, at reduced doses. In some embodiments, the dose is administered by injection, intravenous, or subcutaneous injection, depending in part on whether the administration is transient or chronic. The amount administered can depend on a variety of factors, such as clinical symptoms, patient or subject weight, and whether other medications are being administered. Those skilled in the art will recognize that the route of administration will vary depending on the condition or disease being treated.

可根據需要向對象投予FAP結合劑。投予頻率可由所屬技術領域中具有通常知識者（諸如主治醫師）基於所治療的病況、所治療對象的年齡、所治療病況的嚴重性、所治療對象的一般健康狀態、及類似者的考慮因素來判定。在一些實施例中，一或多次向對象投予有效劑量的FAP結合劑。在一些實施例中，每月一次、少於每月一次（諸如每兩個月或每三個月一次）向對象投予有效劑量的FAP結合劑。在一些實施例中，投予有效劑量的FAP結合劑少於每月一次，諸如每三週一次、每兩週一次、或每週一次。至少一次向對象投予有效劑量的FAP結合劑。在一些實施例中，有效劑量的FAP結合劑可多次投予，包括持續至少一個月、至少六個月、或至少一年的時期。FAP combination therapy may be administered to the recipient as needed. The frequency of administration may be determined by someone with ordinary knowledge in the relevant technical field (such as the attending physician) based on the condition being treated, the age of the recipient, the severity of the condition, the general health status of the recipient, and similar considerations. In some practices, an effective dose of FAP combination therapy may be administered to the recipient once or more. In some practices, an effective dose of FAP combination therapy may be administered to the recipient once a month or less than once a month (e.g., once every two or three months). In some practices, an effective dose of FAP combination therapy may be administered less than once a month, such as once every three weeks, once every two weeks, or once a week. An effective dose of FAP binder shall be administered to the recipient at least once. In some embodiments, the effective dose of FAP binder may be administered multiple times, including for a period of at least one month, at least six months, or at least one year.

在一些實施例中，以有效治療（包括預防）癌症的量投予醫藥組成物。治療有效量一般取決於所治療對象的體重、他或她的身體或健康狀況、所治療病況的範圍、或所治療對象的年齡。適應症In some implementations, pharmaceutical ingredients are administered in doses effective in treating (including preventing) cancer. The effective therapeutic dose generally depends on the patient's weight, his or her physical or health condition, the extent of the condition being treated, or the patient's age. Indications

在一些實施例中，疾病、病症、或病況包括腫瘤疾病。在一些實施例中，至少腫瘤細胞表現FAP。In some embodiments, the disease, symptom, or condition includes oncological diseases. In some embodiments, at least the tumor cells exhibit FAP.

在某些實施例中，腫瘤疾病係實體腫瘤。在一些實施例中，腫瘤疾病包含胃癌、肝癌、肺癌、結腸癌、結腸直腸癌、脾癌、直腸癌、腎癌、乳癌、前列腺癌、皮膚癌、骨癌、血癌、多發性骨髓瘤、神經膠質瘤、卵巢癌、子宮癌、子宮內膜癌、胰臟癌、子宮頸癌、甲狀腺癌、喉癌、急性骨髓性白血病、慢性骨髓性白血病、急性淋巴母細胞白血病、慢性淋巴母細胞白血病、膀胱癌、腦癌、腦腫瘤、神經母細胞瘤、視網膜母細胞瘤、頭頸癌、食道癌、唾液腺癌、及淋巴瘤。癌症的其他實例包括癌、鱗狀細胞癌、淋巴瘤（例如霍奇金氏淋巴瘤及非霍奇金氏淋巴瘤）、胚細胞瘤、肉瘤、及白血病。In some embodiments, the oncological disease is a solid tumor. In some embodiments, the oncological disease includes stomach cancer, liver cancer, lung cancer, colon cancer, colorectal cancer, spleen cancer, rectal cancer, kidney cancer, breast cancer, prostate cancer, skin cancer, bone cancer, leukemia, multiple myeloma, glioma, ovarian cancer, uterine cancer, endometrial cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, bladder cancer, brain cancer, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, esophageal cancer, salivary gland cancer, and lymphoma. Other examples of cancer include carcinoma, squamous cell carcinoma, lymphoma (such as Hodgkin's lymphoma and non-Hodgkin's lymphoma), germ cell tumor, sarcoma, and leukemia.

可使用FAP結合劑（例如抗FAP抗體或其抗原結合片段、雙特異性抗、或其融合蛋白）治療的其他細胞增殖性病症，但不限於位於下列的腫瘤：腹部、骨骼、乳房、消化系統、肝臟、脾臟、胰腺、肺、腹膜、內分泌腺（腎上腺、甲狀旁腺、腦下垂體、睾丸、卵巢、胸腺、甲狀腺）、眼、頭頸、神經系統（中樞及周邊）、淋巴系統、骨盆腔、皮膚、軟組織、脾臟、胸部區域、及泌尿生殖系統。亦包括癌前病況或病變及癌症轉移。在某些實施例中，癌症係選自由下列所組成之群組：腎細胞癌、皮膚癌、肺癌、胰臟癌、結腸直腸癌、乳癌、腦癌、頭頸癌。Other cytoproliferative disorders that can be treated with FAP binding agents (such as anti-FAP antibodies or their antigen-binding fragments, bispecific antibodies, or their fusion proteins), but not limited to tumors located in the following areas: abdomen, bones, breast, digestive system, liver, spleen, pancreas, lungs, peritoneum, endocrine glands (adrenal glands, parathyroid glands, pituitary gland, testes, ovaries, thymus, thyroid glands), eyes, head and neck, nervous system (central and peripheral), lymphatic system, pelvis, skin, soft tissues, spleen, chest region, and genitourinary system. This also includes precancerous conditions or lesions and cancer metastases. In some embodiments, cancer is selected from the following groups: kidney cancer, skin cancer, lung cancer, pancreatic cancer, colorectal cancer, breast cancer, brain cancer, and head and neck cancer.

本揭露提供一種降低腫瘤生長速率或腫瘤細胞數目的方法，其包括使腫瘤細胞與有效量的上述FAP結合劑（例如抗FAP抗體或其抗原結合片段、雙特異性抗體、或其融合蛋白）或醫藥組成物接觸。This disclosure provides a method for reducing tumor growth rate or tumor cell number, comprising contacting tumor cells with an effective amount of the aforementioned FAP binding agent (e.g., an anti-FAP antibody or its antigen-binding fragment, a bispecific antibody, or its fusion protein) or a pharmaceutical composition.

本揭露提供一種殺滅腫瘤細胞之方法，其包括使腫瘤細胞與有效量的上述FAP結合劑（例如抗FAP抗體或其抗原結合片段、雙特異性抗體、或其融合蛋白）或醫藥組成物接觸。 組合治療 This disclosure provides a method for killing tumor cells, comprising contacting the tumor cells with an effective amount of the aforementioned FAP binding agent (e.g., an anti-FAP antibody or its antigen-binding fragment, a bispecific antibody, or its fusion protein) or a pharmaceutical composition. Combination Therapy

本文所提供之FAP結合劑（例如抗FAP抗體或其抗原結合片段、雙特異性抗體、或其融合蛋白）可與一或多種治療劑組合投予。因此，本文提供了用於如本文所述之系統、組成物、及/或治療方法中的組合治療或共治療。應理解，本文所提供之任何組合療法可與本文所提供之任一或多種化合物組合使用，例如與本文所提供之FAP結合劑（例如抗FAP抗體或其抗原結合片段、雙特異性抗體、或其融合蛋白）、其ADC、或包含其的組合療法中之任一者組合使用。The FAP conjugates provided herein (e.g., anti-FAP antibodies or their antigen-binding fragments, bispecific antibodies, or fusion proteins thereof) can be administered in combination with one or more therapeutic agents. Therefore, this document provides for combination therapy or co-treatment in the systems, compositions, and/or treatment methods described herein. It should be understood that any combination therapy provided herein may be used in combination with any one or more compounds provided herein, such as any FAP conjugate (e.g., anti-FAP antibodies or their antigen-binding fragments, bispecific antibodies, or fusion proteins thereof), its ADC, or any combination therapy containing it.

在一些實施例中，本揭露涵蓋的共治療可與一或多種另外的治療活性化合物（包括本文所提供之FAP結合劑）同時、分開、或順序組合投予。任何適當的共治療可用於本揭露的系統、組成物、及/或方法中，且其包括用於治療癌症的共治療。例如，治療劑可包含任何適合所治療的特定適應症的活性成分，較佳地具有不會不利地影響該共治療中的FAP結合劑及/或其他治療劑之互補活性的活性成分。In some embodiments, the co-treatments covered by this disclosure may be administered simultaneously, separately, or sequentially in combination with one or more other therapeutically active compounds, including the FAP conjugates provided herein. Any suitable co-treatment may be used in the systems, compositions, and/or methods of this disclosure, and includes co-treatments for the treatment of cancer. For example, a treatment may contain any active ingredient suitable for the specific indication being treated, preferably having an active ingredient that does not adversely affect the complementary activity of the FAP conjugate and/or other treatments in the co-treatment.

在一些實施例中，另外的治療劑係另一抗癌劑，例如微管破壞劑、抗代謝劑、拓樸異構酶抑制劑、DNA嵌入劑、烷化劑、荷爾蒙療法、激酶抑制劑、受體拮抗劑、腫瘤細胞凋亡活化劑、或抗血管生成劑。在一些實施例中，另外的治療劑係免疫調節劑、細胞生長抑制劑、細胞黏附抑制劑、細胞毒性劑或細胞生長抑制劑、細胞凋亡活化劑、或增加細胞對細胞凋亡誘導劑敏感性的試劑。 免疫調節劑 In some embodiments, the additional treatment is another anticancer agent, such as a microtubule disruptor, antimetabolite, topoisomerase inhibitor, DNA intercalation agent, alkylating agent, hormone therapy, kinase inhibitor, receptor antagonist, tumor cell apoptosis activator, or antiangiogenic agent. In some embodiments, the additional treatment is an immunomodulator, cell growth inhibitor, cell adhesion inhibitor, cytotoxic agent or cell growth inhibitor, apoptosis activator, or a reagent that increases cell sensitivity to apoptosis inducing agents. Immunomotors

可共投予的PD-L1 (CD274)或PD-1 (PDCD1)之抑制劑之實例包括派姆單抗(pembrolizumab)、納武單抗(nivolumab)、西米普利單抗(cemiplimab)、皮地利珠單抗(pidilizumab)、AMP-224、MEDI0680 (AMP-514)、斯巴達珠單抗(spartalizumab)、阿特珠單抗(atezolizumab)、阿維魯單抗(avelumab)、德瓦魯單抗(durvalumab)、BMS-936559、柯希利單抗(cosibelimab) (CK-301)、薩善利單抗(sasanlimab) (PF-06801591)、替雷利珠單抗(tislelizumab) (BGB-A317)、GLS-010 (WBP-3055)、AK-103 (HX-008)、AK-105、CS-1003、HLX-10、瑞弗利單抗(retifanlimab) (MGA-012)、BI-754091、巴斯利單抗(balstilimab) (AGEN-2034)、AMG-404、特瑞普利單抗(toripalimab) (JS-001)、西利單抗(cetrelimab) (JNJ-63723283)、傑諾珠單抗(genolimzumab) (CBT-501)、LZM-009、帕洛利單抗(prolgolimab) (BCD-100)、洛達利單抗(lodapolimab) (LY-3300054)、SHR-1201、卡瑞利珠單抗(camrelizumab) (SHR-1210)、Sym-021、布格利單抗(budigalimab) (ABBV-181)、PD1-PIK、BAT-1306、阿維魯單抗(MSB0010718C)、CX-072、CBT-502、多斯利單抗(dostarlimab) (TSR-042)、MSB-2311、JTX-4014、BGB-A333、SHR-1316、CS-1001 (WBP-3155)、恩弗利單抗(envafolimab) (KN-035)、信迪利單抗(sintilimab) (IBI-308)、HLX-20、KL-A167、STI-A1014、STI-A1015 (IMC-001)、BCD-135、FAZ-053、TQB-2450、MDX1105-01、GS-4224、GS-4416、INCB086550、MAX10181、賽帕利單抗(zimberelimab) (AB122)、斯巴達珠單抗(PDR-001)、及揭示於WO2018195321、WO2020014643、WO2019160882、或WO2018195321中之化合物、以及多特異性抑制劑FPT-155 (CTLA4/PD-L1/CD28)、PF-06936308 (PD-1/ CTLA4)、MGD-013 (PD-1/LAG-3)、FS-118 (LAG-3/PD-L1)、RO-7247669 (PD-1/LAG-3)、MGD-019 (PD-1/CTLA4)、KN-046 (PD-1/CTLA4)、MEDI-5752 (CTLA4/PD-1)、RO-7121661 (PD-1/TIM-3)、RG7769 (PD-1/TIM-3)、TAK-252 (PD-1/OX40L)、XmAb-20717 (PD-1/CTLA4)、AK-104 (CTLA4/PD-1)、FS-118 (LAG-3/PD-L1)、FPT-155 (CTLA4/PD-L1/CD28)、GEN-1046 (PD-L1/4-1BB)、濱他福α (bintrafusp alpha)（M7824；PD-L1/TGFβ-EC域）、CA-170 (PD-L1/VISTA)、CDX-527 (CD27/PD-L1)、LY-3415244 (TIM3/PDL1)、及INBRX-105 (4-1BB/PDL1)。在一些實施例中，PD-L1抑制劑係小分子抑制劑，諸如CA-170、GS-4224、GS-4416、及拉澤替尼(lazertinib) (GNS-1480; PD-L1/EGFR)。Examples of co-administerable PD-L1 (CD274) or PD-1 (PDCD1) inhibitors include pembrolizumab, nivolumab, cemiplimab, pidilizumab, AMP-224, MEDI0680 (AMP-514), spartalizumab, atezolizumab, avelumab, durvalumab, BMS-936559, cosibelimab (CK-301), sasanlimab (PF-06801591), tislelizumab (BGB-A317), and GLS-010. (WBP-3055), AK-103 (HX-008), AK-105, CS-1003, HLX-10, retifanlimab (MGA-012), BI-754091, balstilimab (AGEN-2034), AMG-404, toripalimab (JS-001), cetrelimab (JNJ-63723283), genolimzumab (CBT-501), LZM-009, prolgolimab (BCD-100), lodapolimab (LY-3300054), SHR-1201, camrelizumab (SHR-1210), Sym-021, budigalimab (ABBV-181), PD1-PIK, BAT-1306, averumarubib (MSB0010718C), CX-072, CBT-502, dostarlimab (TSR-042), MSB-2311, JTX-4014, BGB-A333, SHR-1316, CS-1001 (WBP-3155), envafolimab (KN-035), sintilimab (IBI-308), HLX-20, KL-A167, STI-A1014, STI-A1015 (IMC-001), BCD-135, FAZ-053, TQB-2450, MDX1105-01, GS-4224, GS-4416, INCB086550, MAX10181, zimberelimab (AB122), spartazolizumab (PDR-001), and compounds disclosed in WO2018195321, WO2020014643, WO2019160882, or WO2018195321, as well as the multispecific inhibitors FPT-155 (CTLA4/PD-L1/CD28), PF-06936308 (PD-1/CTLA4), MGD-013 (PD-1/LAG-3), FS-118 (LAG-3/PD-L1), RO-7247669 (PD-1/LAG-3), MGD-019 (PD-1/CTLA4), KN-046 (PD-1/CTLA4), MEDI-5752 (CTLA4/PD-1), RO-7121661 (PD-1/TIM-3), RG7769 (PD-1/TIM-3), TAK-252 (PD-1/OX40L), (PD-L1/4-1BB), bintrafusp α) (M7824; PD-L1/TGFβ-EC domain), CA-170 (PD-L1/VISTA), CDX-527 (CD27/PD-L1), LY-3415244 (TIM3/PDL1), and INBRX-105 (4-1BB/PDL1). In some embodiments, the PD-L1 inhibitor is a small molecule inhibitor, such as CA-170, GS-4224, GS-4416, and lazertinib (GNS-1480; PD-L1/EGFR).

可共投予的TIGIT之抑制劑之實例包括替瑞利尤單抗(tiragolumab) (RG-6058)、維博利單抗(vibostolimab)、多伐那利單抗(domvanalimab) (AB154)、AB308、BMS-986207、AGEN-1307、COM-902、或厄提吉利單抗(etigilimab)。Examples of TIGIT inhibitors that can be co-administered include tiragolumab (RG-6058), vibostolimab, domvanalimab (AB154), AB308, BMS-986207, AGEN-1307, COM-902, or etigilimab.

可共投予的CTLA4之抑制劑之實例包括伊匹單抗(ipilimumab)、曲美木單抗(tremelimumab)、BMS-986218、AGEN1181、澤弗利單抗(zalifrelimab) (AGEN1884)、BMS-986249、MK-1308、REGN-4659、ADU-1604、CS-1002（伊匹單抗生物相似藥(biosimilar)）、BCD-145、APL-509、JS-007、BA-3071、ONC-392、AGEN-2041、HBM-4003、JHL-1155、KN-044、CG-0161、ATOR-1144、PBI-5D3H5、BPI-002、以及多特異性抑制劑FPT-155 (CTLA4/PD-L1/CD28)、PF-06936308 (PD-1/ CTLA4)、MGD-019 (PD-1/CTLA4)、KN-046 (PD-1/CTLA4)、MEDI-5752 (CTLA4/PD-1)、XmAb-20717 (PD-1/CTLA4)、及AK-104 (CTLA4/PD-1)。Examples of co-administerable CTLA4 inhibitors include ipilimumab, tremelimumab, BMS-986218, AGEN1181, and zalifrelimab. (AGEN1884), BMS-986249, MK-1308, REGN-4659, ADU-1604, CS-1002 (ipilimumab biosimilar), BCD-145, APL-509, JS-007, BA-3071, ONC-392, AGEN-2041, HBM-4003, JHL-1155, KN-044, CG-0161, ATOR-1144, PBI-5D3H5, BPI-002, and the multispecific inhibitors FPT-155 (CTLA4/PD-L1/CD28), PF-06936308 (PD-1/CTLA4), MGD-019 (PD-1/CTLA4), KN-046 (PD-1/CTLA4), MEDI-5752 (CTLA4/PD-1), XmAb-20717 (PD-1/CTLA4), and AK-104 (CTLA4/PD-1).

調節T細胞(Treg)活性之抑制或Treg除盡可減輕其對抗腫瘤免疫反應之抑制且具有抗癌效應。參見例如Plitas and Rudensky, Annu. Rev. Cancer Biol. (2020) 4:459-77；Tanaka and Sakaguchi, Eur. J. Immunol. (2019) 49:1140-1146。在一些實施例中，本文所提供之式(I)、(Ia)、(Ib)、或(Ic)之化合物或本文所提供之其醫藥上可接受之鹽係與一或多種Treg活性之抑制劑或Treg除盡劑一起投予。Treg抑制或除盡可放大免疫檢查點抑制劑在癌症治療劑中之效應。Inhibition or elimination of T-reg activity can reduce the suppression of antitumor immune responses and has anticancer effects. See, for example, Plitas and Rudensky, Annu. Rev. Cancer Biol. (2020) 4:459-77; Tanaka and Sakaguchi, Eur. J. Immunol. (2019) 49:1140-1146. In some embodiments, compounds of formulas (I), (Ia), (Ib), or (Ic) provided herein, or their pharmaceutically acceptable salts provided herein, are administered co-administered with one or more inhibitors of Treg activity or Treg eliminaters. Treg inhibition or elimination can amplify the efficacy of immune checkpoint inhibitors in cancer therapies.

在一些實施例中，本文所提供之化合物或其醫藥上可接受之鹽係與一或多種Treg抑制劑一起投予。在一些實施例中，Treg抑制劑可抑制Treg至腫瘤微環境中之遷移。在一些實施例中，Treg抑制劑可降低Treg之免疫抑制功能。在一些實施例中，Treg抑制劑可調節細胞表型及誘導促發炎細胞介素之生產。例示性Treg抑制劑包括但不限於CCR4（NCBI基因ID：1233）拮抗劑及下列之降解劑：Ikaros鋅指蛋白（例如Ikaros（IKZF1；NCBI基因ID：10320）、Helios（IKZF2；NCBI基因ID：22807）、Aiolos（IKZF3；NCBI基因ID：22806）、及Eos（IKZF4；NCBI基因ID：64375）。In some embodiments, the compounds provided herein or their pharmaceutically acceptable salts are administered co-administered with one or more Treg inhibitors. In some embodiments, the Treg inhibitors inhibit the migration of Tregs into the tumor microenvironment. In some embodiments, the Treg inhibitors reduce the immunosuppressive function of Tregs. In some embodiments, the Treg inhibitors modulate cell phenotype and induce the production of pro-inflammatory cytokines. Exemplary Treg inhibitors include, but are not limited to, CCR4 (NCBI gene ID: 1233) antagonists and the following degraders: Ikaros zinc finger proteins (e.g., Ikaros (IKZF1; NCBI gene ID: 10320), Helios (IKZF2; NCBI gene ID: 22807), Aiolos (IKZF3; NCBI gene ID: 22806), and Eos (IKZF4; NCBI gene ID: 64375).

可共投予的Helios降解劑之實例包括但不限於I-57 (Novartis)及揭示於WO2019038717、WO2020012334、WO20200117759、WO2021101919、及WO2023178181中之化合物。Examples of co-administerable Helios degraders include, but are not limited to, I-57 (Novartis) and compounds disclosed in WO2019038717, WO2020012334, WO20200117759, WO2021101919 and WO2023178181.

在一些實施例中，本文所提供之化合物或其醫藥上可接受之鹽係與一或多種Treg除盡劑一起投予。在一些實施例中，Treg除盡劑係抗體。在一些實施例中，Treg除盡抗體具有抗體依賴性細胞毒性(ADCC)活性。在一些實施例中，Treg除盡抗體經Fc工程改造以擁有增強之ADCC活性。在一些實施例中，Treg除盡抗體係抗體藥物接合物(ADC)。Treg除盡劑之說明性目標包括但不限於CD25（IL2RA；NCBI基因ID：3559）、CTLA4（CD152；NCBI基因ID：1493）；GITR（TNFRSF18；NCBI基因ID：8784）；4-1BB（CD137；NCBI基因ID：3604）、OX-40（CD134；NCBI基因ID：7293）、LAG3（CD223；NCBI基因ID：3902）、TIGIT（NCBI基因ID：201633）、CCR4（NCBI基因ID：1233）、及CCR8（NCBI基因ID：1237）。In some embodiments, the compounds provided herein or their pharmaceutically acceptable salts are administered co-administered with one or more Treg exhaustants. In some embodiments, the Treg exhaustant is an antibody. In some embodiments, the Treg exhaustant antibody has antibody-dependent cytotoxicity (ADCC) activity. In some embodiments, the Treg exhaustant antibody is Fc-engineered to possess enhanced ADCC activity. In some embodiments, the Treg exhaustant antibody is an antibody-drug conjugate (ADC). Explanatory targets for Treg eradicators include, but are not limited to, CD25 (IL2RA; NCBI gene ID: 3559), CTLA4 (CD152; NCBI gene ID: 1493); GITR (TNFRSF18; NCBI gene ID: 8784); 4-1BB (CD137; NCBI gene ID: 3604), OX-40 (CD134; NCBI gene ID: 7293), LAG3 (CD223; NCBI gene ID: 3902), TIGIT (NCBI gene ID: 201633), CCR4 (NCBI gene ID: 1233), and CCR8 (NCBI gene ID: 1237).

在一些實施例中，可共投予的Treg抑制劑或Treg除盡劑包含選擇性結合至選自由下列所組成之群組的細胞表面受體之抗體或其抗原結合片段：C-C模體趨化因子受體4 (CCR4)、C-C模體趨化因子受體7 (CCR7)、C-C模體趨化因子受體8 (CCR8)、C-X-C模體趨化因子受體4 (CXCR4; CD184)、TNFRSF4 (OX40)、TNFRSF18 (GITR, CD357)、TNFRSF9 (4-1BB, CD137)、細胞毒性T淋巴球相關蛋白4 (CTLA4, CD152)、程式性細胞死亡1 (PDCD1, PD-1)、唾液酸Lewis x (CD15s)、CD27、胞外核苷三磷酸二磷酸水解酶1 (ENTPD1; CD39)、蛋白質酪胺酸磷酸酶受體C型(PTPRC; CD45)、神經細胞黏附分子1 (NCAM1; CD56)、選擇素L (SELL; CD62L)、整合素次單元αE (ITGAE; CD103)、介白素7受體(IL7R; CD127)、CD40配體(CD40LG; CD154)、葉酸受體α (FOLR1)、葉酸受體β (FOLR2)、含富白胺酸重複序列32 (LRRC32; GARP)、IKAROS家族鋅指2 (IKZF2; HELIOS)、可誘導T細胞共刺激(ICOS; CD278)、淋巴球活化3 (LAG3; CD223)、轉形生長因子β1 (TGFB1)、A型肝炎病毒細胞性受體2 (HAVCR2; CD366; TIM3)、具有Ig及ITIM域之T細胞免疫受體(TIGIT)、TNF受體超家族成員1B (CD120b; TNFR2)、IL2RA (CD25)、或其組合。In some embodiments, the co-administerable Treg inhibitor or Treg eradicater comprises an antibody or antigen-binding fragment thereof that selectively binds to a cell surface receptor selected from the following groups: C-C motif chemokine receptor 4 (CCR4), C-C motif chemokine receptor 7 (CCR7), C-C motif chemokine receptor 8 (CCR8), C-X-C motif chemokine receptor 4 (CXCR4; CD184), TNFRSF4 (OX40), TNFRSF18 (GITR, CD357), TNFRSF9 (4-1BB, CD137), cytotoxic T lymphocyte-associated protein 4 (CTLA4, CD152), programmed cell death 1 (PDCD1, PD-1), sialic acid Lewis x (CD15s), CD27, extracellular nucleoside triphosphate diphosphate hydrolase 1 (ENTPD1; CD39), protein tyrosine phosphatase receptor type C (PTPRC; CD45), neural cell adhesion molecule 1 (NCAM1; CD56), selectin L (SELL; CD62L), integrin subunit αE (ITGAE; CD103), interleukin-7 receptor (IL7R; CD127), CD40 ligand (CD40LG; CD154), folate receptor α (FOLR1), folate receptor β (FOLR2), leucine-rich repeat sequence 32 (LRRC32; GARP), IKAROS family zinc finger 2 (IKZF2; HELIOS), inducible T cell co-stimulation (ICOS; CD278), lymphocyte activation 3 (LAG3; CD223), transforming growth factor β1 (TGFB1), hepatitis A virus cytoreceptor 2 (HAVCR2; CD366; TIM3), T cell immune receptor with Ig and ITIM domains (TIGIT), TNF receptor superfamily member 1B (CD120b; TNFR2), IL2RA (CD25), or combinations thereof.

可投予的Treg除盡抗CCR8抗體的實例包括但不限於本文所揭露的參考文獻中所述的抗CCR8抗體，該等文獻之各者的全部內容以引用方式併入本文中。Examples of Tregs that can be administered, excluding anti-CCR8 antibodies, include, but are not limited to, the anti-CCR8 antibodies described in the references disclosed herein, the entire contents of which are incorporated herein by reference.

在一些實施例中，CCR8抗體係具有ADCC活性的單株抗體。此類抗體係所屬技術領域中已知的，例如來自WO2020138489 Al，其以引用方式包括於本文中。在一些實施例中，CCR8抗體係選自WO2020138489 Al中所揭示的抗體，特別係如WO2020138489 Al的請求項中所呈現的抗體。在一些實施例中，CCR8抗體係選自WO2020138489 Al中所揭示的人源化抗體，特別係如WO2020138489 Al的請求項中所呈現的人源化抗體。在一些實施例中，CCR8抗體係來自WO2020138489 Al的抗體10A11、2C7、或19D7或其人源化變體；特別係10A11或其人源化變體；更特別係人源化10A11抗體。在一些實施例中，CCR8抗體係19D7或人源化19D7抗體。In some embodiments, the CCR8 antibody system is a monoclonal antibody with ADCC activity. Such antibody systems are known in the art, for example, from WO2020138489 A1, which is incorporated herein by reference. In some embodiments, the CCR8 antibody system is selected from the antibodies disclosed in WO2020138489 A1, particularly those presented in the claim of WO2020138489 A1. In some embodiments, the CCR8 antibody system is selected from the humanized antibodies disclosed in WO2020138489 A1, particularly those presented in the claim of WO2020138489 A1. In some embodiments, the CCR8 antibody is derived from antibody 10A11, 2C7, or 19D7 or a humanized variant thereof from WO2020138489 A1; particularly 10A11 or a humanized variant thereof; and even more particularly humanized 10A11 antibody. In some embodiments, the CCR8 antibody is 19D7 or humanized 19D7 antibody.

在一些實施例中，CCR8抗體包括BMS-986340 (Bristol Myers Squibb)、LM-108 (LaNova Medicines)、S-531011 (Shionogi)、FPA157 (Five Prime、Amgen)、IPG-7236 (Immunophage Biomedical)、ICP-B05 (InnoCare Pharma Tech)、SRF-114 (Surface Oncology)、HBM1022 (Harbour BioMed)、HFB1011 (HiFiBio)、BAY-3375968 (Bayer)、IO-1 (Oncurious)、ZL-1218 (Zai Lab)、GB2101 (Genor)、PSB-114 (Sound Biologics)、IPG-A05 (Immunophage Biomedical Co Ltd)、PM-1024 (Biotheus Inc、Adimab LLC)、DT-7012 (Domain Therapeutics SA)、BCG-005 (Biocytogen LLC、Liberothera Co Ltd)、GS-1811 (Jounce Therapeutics Inc、Gilead Sciences Inc)、ABBC-514 (AbbieVie Inc)、GNUV-202 (Genuv Inc)、CHS-3318 (Coherus BioSciences Inc)、CTM-033 (Lepu Biopharma)、Integral Molecular Inc的抗CCR8單株抗體、iBio Inc.的抗CCR8抗體、Bristol-Myers Squibb的抗CCR8抗體、iTeos therapeutics的抗CCR8單株抗體、及/或Flanders Institute for Biotechnology VZW及Oncurious NV的抗CCR8療法。In some implementations, CCR8 antibodies include BMS-986340 (Bristol Myers Squibb), LM-108 (LaNova Medicines), S-531011 (Shionogi), FPA157 (Five Prime, Amgen), IPG-7236 (Immunophage Biomedical), ICP-B05 (InnoCare Pharma Tech), SRF-114 (Surface Oncology), HBM1022 (Harbour BioMed), HFB1011 (HiFiBio), BAY-3375968 (Bayer), IO-1 (Oncurious), ZL-1218 (Zai Lab), GB2101 (Genor), PSB-114 (Sound Biologics), IPG-A05 (Immunophage Biomedical Co Ltd), PM-1024 (Biotheus Inc, Adimab LLC), and DT-7012 (Domain Therapeutics). SA), BCG-005 (Biocytogen LLC, Liberothera Co Ltd), GS-1811 (Jounce Therapeutics Inc, Gilead Sciences Inc), ABBC-514 (AbbieVie Inc), GNUV-202 (Genuv Inc), CHS-3318 (Coherus BioSciences Inc), CTM-033 (Lepu Biopharma), anti-CCR8 monoclonal antibodies from Integral Molecular Inc, anti-CCR8 antibodies from iBio Inc., anti-CCR8 antibodies from Bristol-Myers Squibb, anti-CCR8 monoclonal antibodies from iTeos therapeutics, and/or anti-CCR8 therapies from Flanders Institute for Biotechnology VZW and Oncurious NV.

在一些實施例中，CCR8抗體係描述於下列的抗體：WO2022078277、WO2022081718、WO2022000443、WO2022042690、WO2022003156、WO07044756、CN110835371、CN110835374、WO20138489、WO21142002、WO21152186、WO21163064、WO21178749、WO21194942、WO22136649、WO22136650、WO22136647、WO22241034、WO22256563、WO22256559、WO22268192、WO23288241、WO23010054、WO23020621、WO22211046、WO23098888、WO23116880、WO23137466、TW202330599、WO23174396、WO23193732、WO23208182、WO23206350、WO23219147、US11427640B1、US20210277129A1、US20230119066A1、US20230049152A1、US20220403037A1、US10087259B1、WO2013131010A2、WO2021183685A2、US20230176060A1、US20230101029A1、EP4118105A2、US20220389394A1、US20220365091A1、US20210324028A1、EP3589657A1、WO2018112032A1、EP4114862A2、WO2022216965A1、US20230270857A1、EP4232463A2、EP4225373A1、WO2023147488A1、WO2022200303A1、US20190092875A1、EP2656069A2、US6416954B1、WO2023125729A1、US20230160009A1、WO2023076574A1、US20230107291A1、WO2023046156A1、WO2023044402A1、WO2023036246A1、WO2022165260A1、WO2022256628A1、EP3458473B8、EP4090686A2、EP4081548A1、WO2022216702A1、WO2022192457A1、WO2022192895A1、US20220289838A1、EP4041758A1、EP4025255A1、US20220202950A1、EP3994270A1、EP3990476A1、WO2022063194A1、EP3917966A1、WO2021231327A2、EP3908601A1、US11173325B2、EP3793613A1、EP3762421A2、WO2020214718A1、EP2717941B1、US10401357B2、US20200215111A1、EP2652508B1、US9233120B2、US8512701B2、US20200157518A1、EP3554550A1、US20190255107A1、US20190153115A1、EP3471752A1、WO2019036043A2、US20190041389A1、US20180318417A1、US20170218091A1、WO2017011342A1、WO2015183837A1、US20150259418A1、EP2872170A2、WO2015037000A1、EP2654792A2、EP2655415A2、US20130004416A1、EP2337795A2、US20090220486A1、US20090118175A1、EP1948694A2、WO2007005605A2、WO2005010153A2、或WO2002067771A2。In some embodiments, the CCR8 antibody system is described with the following antibodies: WO2022078277, WO2022081718, WO2022000443, WO2022042690, WO2022003156, WO07044756, CN110835371, CN110835374, WO20138489, WO21142002, WO21152186, WO21163064, WO21178749, WO21194942, WO22136649, W O22136650, WO22136647, WO22241034, WO22256563, WO22256559, WO22268192, WO23288241, WO23010054, WO23020621, WO2 2211046, WO23098888, WO23116880, WO23137466, TW202330599, WO23174396, WO23193732, WO23208182, WO23206350, WO23 219147, US11427640B1, US20210277129A1, US20230119066A1, US20230049152A1, US20220403037A1, US10087259B1, WO20 13131010A2, WO2021183685A2, US20230176060A1, US20230101029A1, EP4118105A2, US20220389394A1, US20220365091A1 , US20210324028A1, EP3589657A1, WO2018112032A1, EP4114862A2, WO2022216965A1, US20230270857A1, EP4232463A2, EP 4225373A1, WO2023147488A1, WO2022200303A1, US20190092875A1, EP2656069A2, US6416954B1, WO2023125729A1, US20230 160009A1, WO2023076574A1, US20230107291A1, WO2023046156A1, WO2023044402A1, WO2023036246A1, WO2022165260A1, W O2022256628A1, EP3458473B8, EP4090686A2, EP4081548A1, WO2022216702A1, WO2022192457A1, WO2022192895A1, US2022 0289838A1, EP4041758A1, EP4025255A1, US20220202950A1, EP3994270A1, EP3990476A1, WO2022063194A1, EP3917966A1, WO2021231327A2, EP3908601A1, US11173325B2, EP3793613A1, EP3762421A2, WO2020214718A1, EP2717941B1, US10401357 B2, US20200215111A1, EP2652508B1, US9233120B2, US8512701B2, US20200157518A1, EP3554550A1, US20190255107A1, US 20190153115A1, EP3471752A1, WO2019036043A2, US20190041389A1, US20180318417A1, US20170218091A1, WO2017011342 A1, WO2015183837A1, US20150259418A1, EP2872170A2, WO2015037000A1, EP2654792A2, EP2655415A2, US20130004416A1, EP2337795A2, US20090220486A1, US20090118175A1, EP1948694A2, WO2007005605A2, WO2005010153A2, or WO2002067771A2.

在一些實施例中，CCR8抗體係可從具有ATCC登錄號PTA-6940、PTA-6938、或PTA-6939的融合瘤獲得的抗體。In some embodiments, the CCR8 antibody system can be obtained from fusion tumors with ATCC accession numbers PTA-6940, PTA-6938, or PTA-6939.

在一些實施例中，CCR8抗體係HBM1022抗體，如揭示於Lu et al. HBM1022, a novel anti-CCR8 antibody depletes tumor-infiltrating regulatory T cells via enhanced ADCC activity, mediates potent anti-tumor activity with Keytruda. Journal for ImmunoTherapy of Cancer 2020; 8:doi: 10.1136/jitc-2020-SITC2020.0711。 In some embodiments, the CCR8 antibody is the HBM1022 antibody, as revealed in Lu et al . HBM1022, a novel anti-CCR8 antibody depletes tumor-infiltrating regulatory T cells via enhanced ADCC activity, mediates potent anti-tumor activity with Keytruda. Journal for ImmunoTherapy of Cancer 2020; 8:doi: 10.1136/jitc-2020-SITC2020.0711.

在一些實施例中，CCR8抗體係FPA157抗體，如揭示於Rankin A, Naik E861 Development of FPA157, an anti-CCR8 depleting antibody engineered to preferentially eliminate tumor-infiltrating T regulatory cells. Journal for ImmunoTherapy of Cancer 2020; 8:doi: 10.1136/jitc-2020-SITC2020.0861。In some embodiments, the CCR8 antibody is an FPA157 antibody, as revealed in Rankin A, Naik E861 Development of FPA157, an anti-CCR8 depleting antibody engineered to preferentially eliminate tumor-infiltrating T regulatory cells. Journal for ImmunoTherapy of Cancer 2020; 8:doi: 10.1136/jitc-2020-SITC2020.0861.

在一些實施例中，CCR8抗體係SRFl 14抗體，如揭示於Lake A, Warren M, Das S, et al. Journal for ImmunoTherapy of Cancer 2020; 8:doi: 10.1136/jitc-2020-SITC2020.0726。 In some implementations, the CCR8 antibody is an SRF114 antibody, as revealed in Lake A, Warren M, Das S, et al . Journal for ImmunoTherapy of Cancer 2020; 8:doi: 10.1136/jitc-2020-SITC2020.0726.

在一些實施例中，CCR8抗體係抗CCR8 hlgGl-非岩藻糖基化BMS-986340抗體，如揭示於Lan, Ruth, et al. “Highly selective anti-CCR8 antibody-mediated depletion of regulatory T cells leads to potent antitumor activity alone and in combination with anti-PD-1 in preclinical models.”(2020): 6694-6694及Bayati F, Mohammadi M, Valadi M, Jamshidi S, Foma AM, Sharif-Paghaleh E. The Therapeutic Potential of Regulatory T Cells: Challenges and Opportunities. Front Immunol. 2021;11:585819. Published 2021 Jan 15. doi: 10.3389/fimmu.2020.585819。 In some embodiments, the CCR8 antibody system is against the CCR8 hlgGl-non-fucosylated BMS-986340 antibody, as revealed in Lan, Ruth, et al . “Highly selective anti-CCR8 antibody-mediated depletion of regulatory T cells leads to potent antitumor activity alone and in combination with anti-PD-1 in preclinical models.” (2020): 6694-6694 and Bayati F, Mohammadi M, Valadi M, Jamshidi S, Foma AM, Sharif-Paghaleh E. The Therapeutic Potential of Regulatory T Cells: Challenges and Opportunities. Front Immunol. 2021;11:585819. Published 2021 Jan 15. doi: 10.3389/fimmu.2020.585819.

在一些實施例中，CCR8抗體係奈米抗體，如揭示於Van Damme H, Dombrecht B, Kiss M, Roose H, Allen E, Van Overmeire E, Kancheva D, Martens L, Murgaski A, Bardet PMR, Blancke G, Jans M, Bolli E, Martins MS, Elkrim Y, Dooley J, Boon L, Schwarze JK, Tacke F, Movahedi K, Vandamme N, Neyns B, Ocak S, Scheyltjens I, Vereecke L, Nana FA, Merchiers P, Laoui D, Van Ginderachter JA. Therapeutic depletion of CCR8+ tumor-infiltrating regulatory T cells elicits antitumor immunity and synergizes with anti-PD-1 therapy. J Immunother Cancer.2021 Feb; 9(2):e001749. doi: 10.1136/j itc-2020-001749. PMID: 33589525; PMCID: PMC7887378.In some implementations, CCR8 antibody systems are nanoantibodies, as revealed in Van Damme H, Dombrecht B, Kiss M, Roose H, Allen E, Van Overmeire E, Kancheva D, Martens L, Murgaski A, Bardet PMR, Blancke G, Jans M, Bolli E, Martins MS, Elkrim Y, Dooley J, Boon L, Schwarze JK, Tacke F, Movahedi K, Vandamme N, Neyns B, Ocak S, Scheyltjens I, Vereecke L, Nana FA, Merchiers P, Laoui D, Van Ginderachter JA. Therapeutic depletion of CCR8+ tumor-infiltrating regulatory T cells elicits antitumor immunity and synergizes with anti-PD-1 therapy. J Immunother Cancer. 2021 Feb; 9(2):e001749. doi: 10.1136/j itc-2020-001749. PMID: 33589525; PMCID: PMC7887378.

在一些實施例中，CCR8抗體係azirkitug、cafelkibart、denikitug、lanerkitug。In some implementations, the CCR8 antibody systems are azirkitug, cafekibatar, denikitug, and lanerkitug.

可投予的Treg除盡抗CCR4抗體之實例包括莫格利珠單抗(mogamulizumab)。 化學治療劑 Examples of administerable Tregs that eradicate anti-CCR4 antibodies include mogamulizumab. Chemotherapy

在一些實施例中，本文所提供之化合物係與化學治療劑或抗贅瘤劑一起投予。In some embodiments, the compounds described herein are administered together with chemotherapy agents or antitumor agents.

如本文中所使用，用語「化學治療劑(chemotherapeutic agent/chemotherapeutic)」（或在用化學治療劑治療之情況下之「化學療法(chemotherapy)」）意欲涵蓋可用於治療癌症之任何非蛋白質（例如非肽）化學化合物。化學治療劑之實例包括但不限於：烷化劑，諸如噻替派及環磷醯胺(CYTOXAN ®)；烷基磺酸酯，諸如白消安、英丙舒凡(improsulfan)、及哌泊舒凡(piposulfan)；氮丙啶，諸如苯佐替派(benzodepa)、卡波醌(carboquone)、美妥替呱(meturedepa)、及烏瑞替派(uredepa)；乙烯亞胺及甲基三聚氰胺，包括六甲蜜胺(altretamine)、三乙烯三聚氰胺、三乙烯磷醯胺、三乙烯硫磷醯胺、及三羥甲基三聚氰胺；乙醯精寧(acetogenin)，例如布拉他辛(bullatacin)及布拉他辛酮(bullatacinone)；喜樹鹼，包括合成類似物拓撲替康；苔蘚蟲素、海洋抑素(callystatin)；CC-1065，包括其阿多來新(adozelesin)、卡折來新(carzelesin)、及比折來新(bizelesin)合成類似物；念珠藻素(cryptophycin)，特別是念珠藻素1及念珠藻素8；海兔毒素(dolastatin)；雙聯黴素，包括合成類似物KW-2189及CBI-TMI；艾榴塞洛素(eleutherobin)；5-氮雜胞苷；水鬼蕉鹼(pancratistatin)；沙考地汀(sarcodictyin)；海綿抑素(spongistatin)；氮芥，諸如氯芥苯丁酸、萘氮芥(chlornaphazine)、環磷醯胺、葡磷醯胺(glufosfamide)、伊沃醯胺(evofosfamide)、苯達莫司汀、雌二醇氮芥(estramustine)、異環磷醯胺(ifosfamide)、二氯甲二乙胺(mechlorethamine)、二氯甲二乙胺氧化物鹽酸鹽、美法侖、新恩比興(novembichin)、芬司特瑞(phenesterine)、潑尼氮芥(prednimustine)、曲洛磷胺(trofosfamide)、及尿嘧啶氮芥；亞硝基尿素，諸如卡莫司汀、吡葡亞硝脲(chlorozotocin)、福莫司汀(foremustine)、洛莫司汀、尼氮芥(nimustine)、及雷莫司汀(ranimustine)；抗生素，諸如烯二炔抗生素（例如卡利奇黴素，特別是卡利奇黴素γII及卡利奇黴素phiI1）、達內黴素(dynemicin)，包括達內黴素A、雙膦酸鹽，諸如氯屈膦酸鹽(clodronate)、埃斯培拉黴素(esperamicin)、新抑癌素(neocarzinostatin)發色團及相關色素蛋白烯二炔抗生素發色團、阿克拉黴素(aclacinomycin)、放線菌素、安曲黴素(authramycin)、氮絲胺酸(azaserine)、博來黴素、放線菌素C、卡拉星(carabicin)、卡尼米辛(carrninomycin)、嗜癌素(carzinophilin)、色黴素(chromomycin)、放線菌素D、道諾黴素、地托比星(detorubicin)、6-重氮-5-側氧基-L-正白胺酸、阿黴素（包括N- 啉基-阿黴素、氰基N- 啉基-阿黴素、2-吡咯啉-阿黴素、及去氧阿黴素(deoxydoxorubicin)）、泛艾黴素、依索比星(esorubicin)、艾達黴素、麻西羅黴素(marcellomycin)、絲裂黴素，諸如絲裂黴素C、黴酚酸(mycophenolic acid)、諾加黴素(nogalamycin)、橄欖黴素(olivomycin)、培洛黴素(peplomycin)、泊非黴素(porfiromycin)、嘌呤黴素、三鐵阿黴素(quelamycin)、羅多比星(rodorubicin)、鏈黑黴素(streptonigrin)、鏈脲黴素、殺結核菌素(tubercidin)、鳥苯美司(ubenimex)、淨司他丁(zinostatin)、及佐柔比星(zorubicin)；抗代謝物，諸如胺甲喋呤及5-氟尿嘧啶(5-FU)；葉酸類似物，諸如德莫喋呤(demopterin)、胺甲喋呤、蝶羅呤(pteropterin)、及曲美沙特(trimetrexate)；嘌呤類似物，諸如克拉屈濱、噴司他丁、氟達拉濱、6-巰嘌呤、硫咪嘌呤(thiamiprine)、及硫鳥嘌呤；嘧啶類似物，諸如安西他濱(ancitabine)、阿扎胞苷(azacitidine)、6-硫唑脲嘧啶(6-azauridine)、卡莫氟(carmofur)、阿糖胞苷、二去氧尿苷、去氧氟尿苷(doxifluridine)、依諾他濱(enocitabine)、及氟尿苷；雄激素，諸如卡魯睪酮(calusterone)、丙酸屈他雄酮(dromostanolone propionate)、環硫雄醇(epitiostanol)、美雄烷(mepitiostane)、及睪內酯(testolactone)；抗腎上腺劑，諸如胺魯米特、米托坦、及曲洛司坦(trilostane)；葉酸補充劑，諸如醛葉酸(frolinic acid)；放射治療劑，諸如鐳-223；新月毒素(trichothecene)，特別是T-2毒素、韋拉庫林A (verracurin A)、桿孢菌素A (roridin A)及安奎定(anguidine)；類紫杉醇(taxoid)，諸如太平洋紫杉醇(TAXOL ®)、亞柏杉(abraxane)、多西紫杉醇(TAXOTERE ®)、卡巴他賽、BIND-014、替司他賽(tesetaxel)；薩必沙布林(sabizabulin) (Veru-111)；鉑類似物，諸如順鉑及卡鉑、NC-6004奈鉑(nanoplatin)；醋葡醛內酯(aceglatone)；醛磷醯胺糖苷(aldophosphamide glycoside)；胺基乙醯丙酸(aminolevulinic acid)；恩尿嘧啶(eniluracil)；安吖啶(amsacrine)；赫布西爾(hestrabucil)；比生群(bisantrene)；依達曲沙(edatraxate)；得弗伐胺(defofamine)；地美可辛(demecolcine)；地吖醌(diaziquone)；艾弗欣(elformthine)；依利醋銨(elliptinium acetate)；埃博黴素；依託格魯(etoglucid)；硝酸鎵；羥基脲；蘑菇多糖(lentinan)；亞葉酸(leucovorin)；氯尼達明(lonidamine)；類美坦素(maytansinoid)，諸如美坦素及安絲菌素；米托胍腙(mitoguazone)；米托蒽醌(mitoxantrone)；莫哌達醇(mopidamol)；二胺硝吖啶(nitracrine)；噴司他汀(pentostatin)；凡那明(phenamet)；吡柔比星(pirarubicin)；洛索蒽醌(losoxantrone)；氟嘧啶；醛葉酸(folinic acid)；鬼臼酸(podophyllinic acid)；2-乙基醯肼(2-ethylhydrazide)；丙卡巴肼；多醣-K (PSK)；雷佐生(razoxane)；利索新(rhizoxin)；西索菲蘭(sizofiran)；鍺螺胺(spirogermanium)；細交鏈孢菌酮酸(tenuazonic acid)；曲貝替定(trabectedin)、三亞胺醌(triaziquone)；2,2',2''-三氯三乙胺(trichlorotriemylamine)；胺甲酸酯；長春地辛(vindesine)；達卡巴仁；甘露莫司汀(mannomustine)；二溴甘露醇(mitobronitol)；二溴衛矛醇(mitolactol)；哌泊溴烷(pipobroman)；伽托辛(gacytosine)；阿拉伯糖苷(「Ara-C」)；環磷醯胺；賽派塔(thiopeta)；氯芥苯丁酸；吉西他濱(GEMZAR ®)；6-硫鳥嘌呤；巰嘌呤；胺甲喋呤；長春鹼；鉑；依託泊苷(VP-16)；異環磷醯胺；米托蒽醌(mitroxantrone)；長春新鹼(vancristine)；長春瑞濱(NAVELBINE ®)；諾安托(novantrone)；替尼泊苷；依達曲沙(edatrexate)；道諾黴素(daunomycin)；胺喋呤；希羅達(xeoloda)；伊班膦酸鹽(ibandronate)；CPT-11；拓撲異構酶抑制劑RFS 2000；二氟甲基鳥胺酸(DFMO)；類視黃素，諸如視黃酸；卡培他濱；NUC-1031；FOLFOX（醛葉酸、5-氟尿嘧啶、奧沙利鉑）；FOLFIRI（醛葉酸、5-氟尿嘧啶、伊立替康）；FOLFOXIRI（醛葉酸、5-氟尿嘧啶、奧沙利鉑、伊立替康）、FOLFIRINOX（醛葉酸、5-氟尿嘧啶、伊立替康、奧沙利鉑）、及以上任一者之醫藥上可接受之鹽、酸、或衍生物。此類藥劑可接合至本文所述之抗體或任何靶向劑上，以產生抗體藥物接合物(ADC)或靶向藥物接合物。 As used herein, the term "chemotherapeutic agent/chemotherapeutic" (or "chemotherapy" in the context of chemotherapy) is intended to encompass any non-protein (e.g., non-peptide) chemical compound that can be used to treat cancer. Examples of chemotherapeutic agents include, but are not limited to, alkylating agents, such as thiotepa and cyclophosphamide (CYTOXAN® ). Alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodepa, carboquone, meturedepa, and uredepa; ethyleneimine and methylmelamine, including altretamine, triethylenemelamine, triethylenephosphonium, triethylenethiophosphonium, and trihydroxymethylmelamine; acetogenins, such as bullatacin and bullatacinone; camptothecin, including the synthetic analogue topotecan; bryophyll, callystatin; CC-1065, including its adolexin (ad Synthetic analogs of ozelesin, carzelesin, and bizelesin; cryptophycin, especially cryptophycin 1 and cryptophycin 8; dolastatin; dimethoprims, including synthetic analogs KW-2189 and CBI-TMI; eleutherobin; 5-azacytidine; pancratistatin; sarcodictyin; spongistatin; nitrogen mustard, such as chlorambucil chlorbutazone, chlornaphazine, cyclophosphamide, glufosfamide, evofosfamide, bendamustine, estradiol nitrogen mustard. estramustine), isocyclophosphamide, mechlorethamine, dichloromethyldiethylamine oxide hydrochloride, melphalan, novoembichin, phenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosourea, such as carmustine, chlorozotocin, foremustine, lomustine, nimustine, and ranimustine; antibiotics, such as endothymecides (e.g., calichimycin, especially calichimycin γII and calichimycin phiI1), danonex (dyn) emicins, including danezin A, bisphosphonates such as clodronate, esperamicin, neocarzinostatin chromophores and related pigment proteins, enediyne antibiotic chromophores, aclacinomycin, actinomycin, autramycin, azaserine, bleomycin, actinomycin C, carabicin, carrninomycin, carzinophilin, chromomycin, actinomycin D, daunomycin, detorubicin, 6-diazo-5-sideoxy-L-leucine, doxorubicin (including N- Linoyl-Amycin, Cyano-N- Linyl-aspergillus, 2-pyrrolino-aspergillus, and deoxydoxorubicin, pan-aspergillus, esorubicin, adapalene, marcellomycin, mitomycin, such as mitomycin C, mycophenolic acid Drugs containing citric acid, nogalamycin, olivomycin, peplomycin, pofiromycin, purinemycin, quelamycin, rodorubicin, streptonigrin, streptouremicin, tubercidin, ubenimex, zinostatin, and zorubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues, such as demopterin, methotrexate, and pteroxetine. Pteropterin and trimetrexate; purine analogs, such as cladrolidine, pentostatin, fludarabine, 6-purine, thiamiprine, and thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, doxifluridine, enocitabine, and fluorouridine; androgens, such as calusterone and dromostanolone. Propionate, epitiostanol, mepitiostane, and testolactone; antiadrenergic agents such as ambroxol, mitotan, and trilostane; folic acid supplements such as frolinic acid; radiation therapy agents such as radion-223; trichothecene, especially T-2 toxin, verracurin A, roridin A, and anguidine; taxoids such as TAXOL® , abraxane, and TAXOTERE® . Cabazitaxel, BIND-014, tesetaxel; sabizabulin (Veru-111); platinum analogs, such as cisplatin and carboplatin, NC-6004 nanoplatin; aceglatone; aldophosphamide glycoside; aminolevulinic acid acid); eniluracil; amsacrine; hestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformthine; elliptinium acetate); Ebolamin; Etoglobulin; Gallium nitrate; Hydroxyurea; Lentinan; Leucovorin; Lonidamine; Maytansinoid, such as Maytansin and Anserine; Mitoguazone; Mitoxantrone; Mopidamol; Nitracrine; Pentostatin; Pheanamet; Pirarubicin; Losoxantrone; Fluoropyrimidine; Folate; Podophyllinic acid; 2-Ethylhydrazide; Procarbazine; Polysaccharide-K (PSK); Razoxane; Rhizoxin; Sizofiran; Spirogermanium; Tenuazonic acid; Trabectedin; Triaziquone; Trichlorotriemylamine; Carbamate; Vindesine; Dacarbazine; Mannomustine; Mitobronitol; Mitolactalol; Pipobroman; Gacytosine; Ara-C; Cyclophosphamide; Thiopeta; Chlorisuccinate; Gemzar® ); 6-Thioguanine; guanine; methotrexate; vincristine; platinum; etoposide (VP-16); isocyclophosphamide; mitroxantrone; vancristine; vinorelbine ( NAVELBINE® ); novantrone; teniposide; edatrexate; daunomycin; methotrexate; xeoloda; ibandronate; CPT-11; topaz isomerase inhibitor RFS 2000; difluoromethylguanosine (DFMO); retinoids, such as retinoic acid; capecitabine; NUC-1031; FOLFOX (folate, 5-fluorouracil, oxaliplatin); FOLFIRI (folate, 5-fluorouracil, irinotecan); FOLFOXIRI (folate, 5-fluorouracil, oxaliplatin, irinotecan), FOLFIRINOX (folate, 5-fluorouracil, irinotecan, oxaliplatin), and pharmaceutically acceptable salts, acids, or derivatives of any of the above. These agents can be conjugated to the antibodies or any targeted agents described herein to produce antibody-drug conjugates (ADCs) or targeted drug conjugates.

在一些實施例中，可共投予的ADC包括貝蘭單抗莫福汀、本妥昔單抗維多汀、卡米達努單抗特西林、曲妥珠單抗德魯替康、曲妥珠單抗恩他新、米爾唯土西單抗索星、拉迪拉妥珠單抗維多汀、隆卡妥昔單抗特西林、薩西土珠單抗戈維特坎、達妥伯單抗德魯替康(DS-1062; Dato-DXd)、英妥珠單抗奧唑米星、吉妥珠單抗奧唑米星、隆卡妥昔單抗特西林、貝蘭單抗莫福汀、米爾唯土西單抗索星、莫昔土莫單抗帕蘇多托克斯、帕特里妥單抗德魯替康、保納珠單抗維多汀、烏比妥單抗利爾索多汀、羅伐匹妥單抗特西林、因福土單抗維多汀、替索妥單抗維多汀、圖薩米單抗拉萬替辛、迪西妥單抗維多汀、泰利妥珠單抗維多汀(ABBV-399)。 細胞毒性劑或細胞生長抑制劑 In some implementations, co-administered ADCs include belanumab mofostatin, bentoximab vedoltin, carmidanumab tecillin, trastuzumab deruticotin, trastuzumab entathine, mirabilite sucralose, radix vedoltin, lonca tuzumab tecillin, sacralite sucralose glevitamin, and datobetab deruticotin (DS-1062). Dato-DXd), Intuzumab (Ozolmicin), Gemtuzumab (Ozolmicin), Loccatusumab (Texacillin), Belanumab (Mofotin), Milvitumab (Sorcin), Moxitomosumab (Pasudotropux), Patrituximab (Delutec), Pannazumab (Vidodine), Urbituximab (Lisoldotin), Rovapitumab (Texacillin), Informumab (Vidodine), Texazumab (Vidodine), Tusamizumab (Vidodine), Dictetuzumab (Vidodine), Texazumab (Vidodine) (ABBV-399). Cytotoxic agents or cell growth inhibitors

在一些實施例中，共治療係細胞毒性劑或細胞生長抑制劑。細胞毒性劑或細胞生長抑制劑的實例包括本文所述之化學治療劑及細胞裂解劑。在一些實施例中，共治療係抗體藥物接合物(ADC)。在一些實施例中，ADC的抗體部份係薩西土珠單抗，其中抗體部份靶向TROP2，且藥物部份係戈維特坎。在一些實施例中，ADC的抗體部份係替索妥單抗，且藥物部份係維多汀組織因子。在一些實施例中，ADC的抗體部份係因福土單抗，且藥物部份係維多汀Nectin4。在一些實施例中，ADC之抗體部份係本妥昔單抗，且藥物部份係維多汀CD30。在一些實施例中，ADC的抗體部份係曲妥珠單抗，且藥物部份係德魯替康HER2。在一些實施例中，ADC之抗體部份係曲妥珠單抗，且藥物部份係恩他新HER2。在一些實施例中，ADC之抗體部份係保納珠單抗，且藥物部份係維多汀CD79。在一些實施例中，ADC之抗體部份係英妥珠單抗，且藥物部份係奧唑米星CD22。在一些實施例中，ADC之抗體部份係吉妥珠單抗，且藥物部份係奧唑米星CD33。在一些實施例中，ADC之抗體部份係隆卡妥昔單抗，且藥物部份係特西林CD19。在一些實施例中，ADC之抗體部份係貝蘭單抗，且藥物部份係莫福汀BCMA。在一些實施例中，ADC的之抗體部份係米爾唯土西單抗，且藥物部份係索星FR⍺。在一些實施例中，ADC之抗體部份係莫昔土莫單抗，且藥物部份係帕蘇多托克斯CD22。 例示性組合療法 乳癌組合療法 In some embodiments, the co-treatment is a cytotoxic agent or a cell growth inhibitor. Examples of cytotoxic agents or cell growth inhibitors include the chemotherapeutic agents and cell lysing agents described herein. In some embodiments, the co-treatment is an antibody-drug conjugate (ADC). In some embodiments, the antibody portion of the ADC is saxituzumab, which targets TROP2, and the drug portion is gavitecan. In some embodiments, the antibody portion of the ADC is texotuximab, and the drug portion is vitolamine tissue factor. In some embodiments, the antibody portion of the ADC is infotumab, and the drug portion is vitolamine Nectin4. In some embodiments, the antibody portion of the ADC is trentoximab, and the drug portion is vitoline CD30. In some embodiments, the antibody portion of the ADC is trastuzumab, and the drug portion is derunotecan HER2. In some embodiments, the antibody portion of the ADC is trastuzumab, and the drug portion is entazoline HER2. In some embodiments, the antibody portion of the ADC is bulazacillin, and the drug portion is vitoline CD79. In some embodiments, the antibody portion of the ADC is intuzumab, and the drug portion is oxozamicin CD22. In some embodiments, the antibody portion of the ADC is gemtruzumab, and the drug portion is oxozamicin CD33. In some embodiments, the antibody portion of the ADC is loncaituximab, and the drug portion is tecillin CD19. In some embodiments, the antibody portion of the ADC is belanumab, and the drug portion is mofostatin (BCMA). In some embodiments, the antibody portion of the ADC is miloxacillin, and the drug portion is sucrazine (FR⍺). In some embodiments, the antibody portion of the ADC is moxitotumumab, and the drug portion is persudotrox (CD22). Exemplary Combination Therapy Breast Cancer Combination Therapy

用以治療乳癌之治療劑包括白蛋白結合型太平洋紫杉醇、阿那曲唑、阿特珠單抗、卡培他濱、卡鉑、順鉑、環磷醯胺、多西紫杉醇、阿黴素、泛艾黴素、依維莫司、依西美坦、氟尿嘧啶、氟維司群、吉西他濱、伊沙匹隆、拉帕替尼、來曲唑、胺甲喋呤、米托蒽醌、太平洋紫杉醇、聚乙二醇化微脂體阿黴素、帕妥珠單抗、泰莫西芬、托瑞米芬、曲妥珠單抗、長春瑞濱、及其任何組合。在一些實施例中，用以治療乳癌（例如HR+/-/HER2 +/-）之治療劑包括曲妥珠單抗(Herceptin ®)、帕妥珠單抗(Perjeta ®)、多西紫杉醇、卡鉑、帕博西尼(Ibrance ®)、來曲唑、曲妥珠單抗恩他新(Kadcyla ®)、氟維司群(Faslodex ®)、奧拉帕尼(Lynparza ®)、艾日布林、圖卡替尼、卡培他濱、拉帕替尼、依維莫司(Afinitor ®)、依西美坦、艾日布林甲磺酸酯(Halaven ®)、及其組合。在一些實施例中，用以治療乳癌之治療劑包括曲妥珠單抗+帕妥珠單抗+多西紫杉醇、曲妥珠單抗+帕妥珠單抗+多西紫杉醇+卡鉑、帕博西尼+來曲唑、圖卡替尼+卡培他濱、拉帕替尼+卡培他濱、帕博西尼+氟維司群、或依維莫司+依西美坦。在一些實施例中，用以治療乳癌之治療劑包括曲妥珠單抗德魯替康(Enhertu ®)、達妥伯單抗德魯替康(DS-1062)、因福土單抗維多汀(Padcev ®)、巴沙福泰(balixafortide)、艾拉司群(elacestrant)、或其組合。在一些實施例中，用以治療乳癌之治療劑包括巴沙福泰+艾日布林。 三陰性乳癌(TNBC)組合療法 Treatments for breast cancer include albumin-bound paclitaxel, anastrozole, atezolizumab, capecitabine, carboplatin, cisplatin, cyclophosphamide, docetaxel, doxorubicin, panemexin, everolimus, exemestane, fluorouracil, fulvestrant, gemcitabine, ixaprilone, lapatinib, letrozole, methotrexate, mitoxantrone, paclitaxel, pegylated liposomal doxorubicin, pertuzumab, temoxifen, toremifene, trastuzumab, vinorelbine, and any combination thereof. In some embodiments, treatments for breast cancer (e.g., HR+/-/HER2 +/-) include trastuzumab ( Herceptin® ), pertuzumab (Perjeta®), docetaxel, carboplatin, palbociclib ( Ibrance® ), letrozole, trastuzumab entacin ( Kadcyla® ), fulvestrant ( Faslodex® ), olaparib ( Lynparza® ), eribulin, tucatinib, capecitabine, lapatinib, everolimus ( Afinitor® ), exemestane, eribulin mesylate ( Halaven® ), and combinations thereof . In some embodiments, treatments for breast cancer include trastuzumab + pertuzumab + docetaxel, trastuzumab + pertuzumab + docetaxel + carboplatin, palbociclib + letrozole, tucatinib + capecitabine, lapatinib + capecitabine, palbociclib + fulvestrant, or everolimus + exemestane. In some embodiments, treatments for breast cancer include trastuzumab ( enhertu® ), datobacillus (DS-1062), informumab ( padcev® ), balixafortide, elacestrant, or combinations thereof. In some practices, treatments for breast cancer include balsamicin and eribulin. Triple-negative breast cancer (TNBC) combination therapy.

用以治療TNBC之治療劑包括阿特珠單抗、環磷醯胺、多西紫杉醇、阿黴素、泛艾黴素、氟尿嘧啶、太平洋紫杉醇、及其組合。在一些實施例中，用以治療TNBC之治療劑包括奧拉帕尼(Lynparza ®)、阿特珠單抗(Tecentriq ®)、太平洋紫杉醇(Abraxane ®)、艾日布林、貝伐單抗(Avastin ®)、卡鉑、吉西他濱、艾日布林甲磺酸酯(Halaven ®)、薩西土珠單抗戈維特坎(Trodelvy ®)、派姆單抗(Keytruda ®)、順鉑、阿黴素、泛艾黴素、或其組合。在一些實施例中，治療TNBC之治療劑包括阿特珠單抗+太平洋紫杉醇、貝伐單抗+太平洋紫杉醇、卡鉑+太平洋紫杉醇、卡鉑+吉西他濱、或太平洋紫杉醇+吉西他濱。在一些實施例中，用以治療TNBC之治療劑包括艾利亞斯酶(eryaspase)、卡瓦替布、艾培昔布、蘆卡帕尼+納武單抗、阿索盧單抗(atezolumab) +太平洋紫杉醇+吉西他濱+卡培他濱+卡鉑、伊巴替布(ipatasertib) +太平洋紫杉醇、拉迪朗妥珠單抗維多汀+派伯利單抗(pembrolimab)、德瓦魯單抗+ DS-8201a、曲拉西利+吉西他濱+卡鉑。在一些實施例中，用以治療TNBC之治療劑包括曲妥珠單抗德魯替康(Enhertu ®)、達妥伯單抗德魯替康(DS-1062)、因福土單抗維多汀(Padcev ®)、巴沙福泰、阿達洛德西莫林(adagloxad simolenin)、萊尼哌嗎-s (NeuVax ®)、納武單抗(Opdivo ®)、蘆卡帕尼、特瑞普利單抗(Tuoyi ®)、卡瑞利珠單抗、卡瓦替布、德瓦魯單抗(Imfinzi ®)、及其組合。在一些實施例中，用以治療TNBC之治療劑包括納武單抗+蘆卡帕尼、貝伐單抗(Avastin ®) +化學療法、特瑞普利單抗+太平洋紫杉醇、特瑞普利單抗+白蛋白結合型太平洋紫杉醇、卡瑞利珠單抗+化學療法、派姆單抗+化學療法、巴沙福泰+艾日布林、德瓦魯單抗+曲妥珠單抗德魯替康、德瓦魯單抗+太平洋紫杉醇、或卡瓦替布+太平洋紫杉醇。 膀胱癌組合療法 Treatments for TNBC include atezolizumab, cyclophosphamide, docetaxel, doxorubicin, ubiquitin, fluorouracil, paclitaxel, and combinations thereof. In some practices, treatments for TNBC include olaparib ( Lynparza® ), atezolizumab (Tecentriq®), paclitaxel ( Abraxane® ), eribulin, bevacizumab ( Avastin® ), carboplatin, gemcitabine, eribulin mesylate ( Halaven® ), sasitumumab-gauvitcan ( Trodelvy® ), pembrolizumab ( Keytruda® ), cisplatin, doxorubicin, ubiquitin, or combinations thereof . In some practices, treatments for TNBC include atezolizumab + paclitaxel, bevacizumab + paclitaxel, carboplatin + paclitaxel, carboplatin + gemcitabine, or paclitaxel + gemcitabine. In other practices, treatments for TNBC include eryaspase, cavotitab, ipexib, descapab + nivolumab, atezolizumab + paclitaxel + gemcitabine + capecitabine + carboplatin, ipatasertib + paclitaxel, radix lantocilizumab + vedotin + pembrolimab, durvalumab + DS-8201a, and triamcinolone + gemcitabine + carboplatin. In some practices, treatments for TNBC include trastuzumab ( enhertu® ), datobacillus (DS-1062), infotumab ( Padcev® ), basafate, adagloxad simolenin, lenipexoles ( NeuVax® ), nivolumab ( Opdivo® ), rucapranib, toripalimab ( Tuoyi® ), camrelizumab, calvatibub, devalumab ( Imfinzi® ), and combinations thereof. In some practices, treatments for TNBC include nivolumab + decapapramib, bevacizumab ( Avastin® ) + chemotherapy, toripalimab + paclitaxel, toripalimab + albumin-bound paclitaxel, camrelizumab + chemotherapy, pembrolizumab + chemotherapy, balsaformide + eribulin, durvalumab + trastuzumab/delutec, durvalumab + paclitaxel, or cavatibub + paclitaxel. Combination therapy for bladder cancer.

用以治療膀胱癌之治療劑包括達妥伯單抗德魯替康(DS-1062)、曲妥珠單抗德魯替康(Enhertu ®)、厄達替尼、依格利昔(eganelisib)、樂伐替尼、貝培阿地白介素(bempegaldesleukin) (NKTR-214)、或其組合。在一些實施例中，用以治療膀胱癌之治療劑包括依格利昔+納武單抗、派姆單抗(Keytruda ®) +因福土單抗維多汀(Padcev ®)、納武單抗+伊匹單抗、度伐魯單抗(duravalumab) +曲美木單抗、樂伐替尼+派姆單抗、因福土單抗維多汀(Padcev ®) +派姆單抗、及貝培阿地白介素+納武單抗。 結腸直腸癌(CRC)組合療法 Treatments for bladder cancer include datobetab (delutecan, DS-1062), trastuzumab (delutecan, Enhertu® ), erdatinib, eganelisib, lenvatinib, beempegaldesleukin (NKTR-214), or combinations thereof. In some practices, treatments for bladder cancer include eganelisib + nivolumab, pembrolizumab ( Keytruda® ) + infutuzumab + vedolfin ( Padcev® ), nivolumab + ipilimumab, duravalumab + trimelimumab, lenvatinib + pembrolizumab, infutuzumab + vedolfin ( Padcev® ) + pembrolizumab, and beempegaldesleukin + nivolumab. Combination therapy for colorectal cancer (CRC)

用以治療CRC之治療劑，包括貝伐單抗、卡培他濱、西妥昔單抗、氟尿嘧啶、伊立替康、亞葉酸、奧沙利鉑、帕尼單抗、ziv-阿柏西普、及其任何組合。在一些實施例中，用以治療CRC之治療劑包括貝伐單抗(Avastin ®)、亞葉酸、5-FU、奧沙利鉑(FOLFOX)、派姆單抗(Keytruda ®)、FOLFIRI、瑞戈非尼(Stivarga ®)、阿柏西普(Zaltrap ®)、西妥昔單抗(Erbitux ®)、朗斯弗(Orcantas ®)、XELOX、FOLFOXIRI、或其組合。在一些實施例中，用以治療CRC之治療劑包括貝伐單抗+亞葉酸+ 5-FU +奧沙利鉑(FOLFOX)、貝伐單抗+ FOLFIRI、貝伐單抗+ FOLFOX、阿柏西普+ FOLFIRI、西妥昔單抗+ FOLFIRI、貝伐單抗+ XELOX、及貝伐單抗+ FOLFOXIRI。在一些實施例中，用以治療CRC之治療劑包括畢尼替尼+康奈非尼(encorafenib) +西妥昔單抗、曲美替尼+達拉菲尼+帕尼單抗、曲妥珠單抗+帕妥珠單抗、那帕布新+ FOLFIRI +貝伐單抗、納武單抗+伊匹單抗。 食道癌及食道胃接合處癌組合療法 Treatments for CRC include bevacizumab, capecitabine, cetuximab, fluorouracil, irinotecan, leucovorin, oxaliplatin, panitumumab, ziv-aflibercept, and any combination thereof. In some practices, treatments for CRC include bevacizumab ( Avastin® ), leucovorin, 5-FU, oxaliplatin (FOLFOX), pembrolizumab ( Keytruda® ), FOLFIRI, regorafenib ( Stivarga® ), aflibercept ( Zaltrap® ), cetuximab ( Erbitux® ), orcantas® , XELOX, fOLFOXIRI, or combinations thereof. In some practices, treatments for CRC include bevacizumab + leucovorin + 5-FU + oxaliplatin (FOLFOX), bevacizumab + FOLFIRI, bevacizumab + FOLFOX, aflibercept + FOLFIRI, cetuximab + FOLFIRI, bevacizumab + XELOX, and bevacizumab + FOLFOXIRI. In other practices, treatments for CRC include binitinib + encorafenib + cetuximab, trametinib + dabrafenib + panitumumab, trastuzumab + pertuzumab, napabuxin + FOLFIRI + bevacizumab, and nivolumab + ipilimumab. Combination therapy for esophageal cancer and esophagogastric junction cancer.

用以治療食道癌及食道胃接合處癌之治療劑包括卡培他濱、卡鉑、順鉑、多西紫杉醇、泛艾黴素、氟嘧啶、氟尿嘧啶、伊立替康、亞葉酸、奧沙利鉑、太平洋紫杉醇、雷莫蘆單抗、曲妥珠單抗、及其任何組合。在一些實施例中，用以治療胃食道接合處癌(GEJ)之治療劑包括賀癌平(herceptin)、順鉑、5-FU、拉米庫單抗(ramicurimab)、或太平洋紫杉醇。在一些實施例中，用以治療GEJ癌症之治療劑包括ALX-148、AO-176、或IBI-188。 胃癌組合療法 Treatments for esophageal cancer and esophagogastric junction cancer include capecitabine, carboplatin, cisplatin, docetaxel, panemifazole, fluoropyrimidine, fluorouracil, irinotecan, leucovorin, oxaliplatin, paclitaxel, ramorumab, trastuzumab, and any combination thereof. In some practices, treatments for gastroesophageal junction cancer (GEJ) include herceptin, cisplatin, 5-FU, ramicurimab, or paclitaxel. In some practices, treatments for GEJ cancer include ALX-148, AO-176, or IBI-188. Gastric Cancer Combination Therapy

用以治療胃癌之治療劑包括卡培他濱、卡鉑、順鉑、多西紫杉醇、泛艾黴素、氟嘧啶、氟尿嘧啶、伊立替康、亞葉酸、絲裂黴素、奧沙利鉑、太平洋紫杉醇、雷莫蘆單抗、曲妥珠單抗、及其任何組合。 頭頸癌組合療法 Treatments for stomach cancer include capecitabine, carboplatin, cisplatin, docetaxel, panemmycin, fluorouracil, fluorouracil, irinotecan, leucovorin, mitomycin, oxaliplatin, paclitaxel, ramorumab, trastuzumab, and any combination thereof. Head and Neck Cancer Combination Therapy

用以治療頭頸癌之治療劑包括阿法替尼、博來黴素、卡培他濱、卡鉑、西妥昔單抗、順鉑、多西紫杉醇、氟尿嘧啶、吉西他濱、羥基脲、胺甲喋呤、納武單抗、太平洋紫杉醇、派姆單抗、長春瑞濱、及其任何組合。Treatments for head and neck cancer include afatinib, bleomycin, capecitabine, carboplatin, cetuximab, cisplatin, docetaxel, fluorouracil, gemcitabine, hydroxyurea, methotrexate, nivolumab, paclitaxel, pembrolizumab, vinorelbine, and any combination thereof.

用以治療頭頸部鱗狀細胞癌(head and neck squamous cell carcinoma, HNSCC)之治療劑包括派姆單抗、卡鉑、5-FU、多西紫杉醇、西妥昔單抗(Erbitux ®)、順鉑、納武單抗(Opdivo ®)、及其組合。在一些實施例中，用以治療HNSCC之治療劑包括派姆單抗+卡鉑+ 5-FU、西妥昔單抗+順鉑+ 5-FU、西妥昔單抗+卡鉑+ 5-FU、順鉑+ 5-FU、及卡鉑+ 5-FU。在一些實施例中，用以治療HNSCC之治療劑包括德瓦魯單抗、德瓦魯單抗+曲美木單抗、納武單抗+伊匹單抗、羅伐盧賽爾(rovaluecel)、派姆單抗、派姆單抗+依波斯他、GSK3359609 +派姆單抗、樂伐替尼+派姆單抗、瑞弗利單抗、瑞弗利單抗+恩諾必單抗(enobituzumab)、ADU-S100 +派姆單抗、依波斯他+納武單抗+伊匹單抗/立魯單抗。 非小細胞肺癌組合療法 Treatments for head and neck squamous cell carcinoma (HNSCC) include pembrolizumab, carboplatin, 5-FU, docetaxel, cetuximab ( Erbitux® ), cisplatin, nivolumab ( Opdivo® ), and combinations thereof. In some practices, treatments for HNSCC include pembrolizumab + carboplatin + 5-FU, cetuximab + cisplatin + 5-FU, cetuximab + carboplatin + 5-FU, cisplatin + 5-FU, and carboplatin + 5-FU. In some practices, treatments for HNSCC include devalumab, devalumab + trimemumab, nivolumab + ipilimumab, rovaluecel, pembrolizumab, pembrolizumab + epipodil, GSK3359609 + pembrolizumab, lenvatinib + pembrolizumab, rivab, rivab + ennobituzumab, ADU-S100 + pembrolizumab, and epipodil + nivolumab + ipilimumab/rivalumab. Combination therapy for non-small cell lung cancer.

用以治療非小細胞肺癌(NSCLC)之治療劑包括阿法替尼、白蛋白結合型太平洋紫杉醇、艾樂替尼、阿特珠單抗、貝伐單抗、貝伐單抗、卡博替尼、卡鉑、順鉑、克唑替尼(crizotinib)、達拉菲尼、多西紫杉醇、埃羅替尼、依託泊苷、吉西他濱、納武單抗、太平洋紫杉醇、派姆單抗、培美曲塞、雷莫蘆單抗、曲美替尼、曲妥珠單抗、凡德他尼、維羅非尼、長春鹼、長春瑞濱、及其任何組合。在一些實施例中，用以治療NSCLC之治療劑包括艾樂替尼(Alecensa ®)、達拉非尼(Tafinlar ®)、曲美替尼(Mekinist ®)、奧希替尼(Tagrisso ®)、恩曲替尼(Tarceva ®)、克唑替尼(Xalkori ®)、派姆單抗(Keytruda ®)、卡鉑、培美曲塞(Alimta ®)、白蛋白結合型太平洋紫杉醇(Abraxane ®)、雷莫蘆單抗(Cyramza ®)、多西紫杉醇、貝伐單抗(Avastin ®)、布格替尼、吉西他濱、順鉑、阿法替尼(Gilotrif ®)、納武單抗(Opdivo ®)、吉非替尼(Iressa ®)、及其組合。在一些實施例中，用以治療NSCLC之治療劑包括達拉菲尼+曲美替尼、派姆單抗+卡鉑+培美曲塞、派姆單抗+卡鉑+白蛋白結合型太平洋紫杉醇、雷莫蘆單抗+多西紫杉醇、貝伐單抗+卡鉑+培美曲塞、派姆單抗+培美曲塞+卡鉑、順鉑+培美曲塞、貝伐單抗+卡鉑+白蛋白結合型太平洋紫杉醇、順鉑+吉西他濱、納武單抗+多西紫杉醇、卡鉑+培美曲塞、卡鉑+白蛋白結合型太平洋紫杉醇、或培美曲塞+順鉑+卡鉑。在一些實施例中，用以NSCLC之治療劑包括達妥伯單抗德魯替康(DS-1062)、曲妥珠單抗德魯替康(Enhertu ®)、因福土單抗維多汀(Padcev ®)、德瓦魯單抗、卡那單抗、西米普利單抗、諾格介白素α、阿維魯單抗、替瑞利尤單抗、多伐那利單抗、維博利單抗、奧西伯利單抗、或其組合。在一些實施例中，用以治療NSCLC之治療劑包括達妥伯單抗德魯替康+派姆單抗、達妥伯單抗德魯替康+德瓦魯單抗、德瓦魯單抗+曲美木單抗、派姆單抗+樂伐替尼+培美曲塞、派姆單抗+奧拉帕尼、諾格介白素α (N-803) +派姆單抗、替瑞利尤單抗+阿特珠單抗、維博利單抗+派姆單抗、或奧西伯利單抗+替雷利珠單抗。 小細胞肺癌組合療法 Treatments for non-small cell lung cancer (NSCLC) include afatinib, albumin-bound paclitaxel, alectinib, atezolizumab, bevacizumab, cabozantinib, carboplatin, cisplatin, crizotinib, dabrafenib, docetaxel, erlotinib, etoposide, gemcitabine, nivolumab, paclitaxel, pembrolizumab, pemetrexed, ramorumab, trametinib, trastuzumab, vandetanib, vemurafenib, vinorelbine, vinorelbine, and any combination thereof. In some practices, treatments for NSCLC include alectinib ( Alecensa® ), dabrafenib (Tafinlar®), trametinib (Mekinist®), osimertinib ( Tagrisso® ), entrectinib ( Tarceva® ) , crizotinib (Xalkori®), pembrolizumab ( Keytruda® ), carboplatin, pemetrexed ( Alimta® ), albumin-bound paclitaxel ( Abraxane® ), ramucirumab ( Cyramza® ) , docetaxel, bevacizumab ( Avastin® ), brigatinib, gemcitabine, cisplatin, afatinib ( Gilotrif® ), nivolumab ( Opdivo® ), gefitinib ( Iressa® ), and combinations thereof . In some practices, treatments for NSCLC include dabrafenib + trametinib, pembrolizumab + carboplatin + pemetrexed, pembrolizumab + carboplatin + albumin-bound paclitaxel, ramorumab + docetaxel, bevacizumab + carboplatin + pemetrexed, pembrolizumab + pemetrexed + carboplatin, cisplatin + pemetrexed, bevacizumab + carboplatin + albumin-bound paclitaxel, cisplatin + gemcitabine, nivolumab + docetaxel, carboplatin + pemetrexed, carboplatin + albumin-bound paclitaxel, or pemetrexed + cisplatin + carboplatin. In some embodiments, treatments for NSCLC include dattabacitraca (DS-1062), trastuzumab ( Enhertu® ), infotiam ( Padcev® ), durvalumab, cannabidiol, cimiprimab, noglin-alpha, averrucumab, tireliumab, dovanalimumab, vimbrolizumab, osimertinib, or combinations thereof. In some practices, treatments for NSCLC include dattatumab/delutecan + pembrolizumab, dattatumab/delutecan + durvalumab, durvalumab + trimemumab, pembrolizumab + lenvatinib + pemetrexed, pembrolizumab + olaparib, noglin-803 (N-803) + pembrolizumab, tislelizumab + atezolizumab, vimbrolizumab + pembrolizumab, or osimertinib + tislelizumab. Combination therapy for small cell lung cancer.

用以治療小細胞肺癌(SCLC)之治療劑包括阿特珠單抗、苯達莫司汀(bendamustime)、卡鉑、順鉑、環磷醯胺、多西紫杉醇、阿黴素、依託泊苷、吉西他濱、伊匹單抗(ipillimumab)、伊立替康、納武單抗、太平洋紫杉醇、替莫唑胺、拓撲替康、長春新鹼、長春瑞濱、及其任何組合。在一些實施例中，用以治療SCLC之治療劑包括阿特珠單抗、卡鉑、順鉑、依託泊苷、太平洋紫杉醇、拓撲替康、納武單抗、德瓦魯單抗、曲拉西利、或其組合。在一些實施例中，用以治療SCLC之治療劑包括阿特珠單抗+卡鉑+依託泊苷、阿特珠單抗+卡鉑、阿特珠單抗+依託泊苷、或卡鉑+太平洋紫杉醇。 卵巢癌組合療法 Treatments for small cell lung cancer (SCLC) include atezolizumab, bendamustime, carboplatin, cisplatin, cyclophosphamide, docetaxel, doxorubicin, etoposide, gemcitabine, ipilimumab, irinotecan, nivolumab, paclitaxel, temozolomide, topotecan, vincristine, vinorelbine, and any combination thereof. In some practices, treatments for SCLC include atezolizumab, carboplatin, cisplatin, etoposide, paclitaxel, topotecan, nivolumab, durvalumab, triprazide, or combinations thereof. In some practices, treatments for SCLC include atezolizumab + carboplatin + etoposide, atezolizumab + carboplatin, atezolizumab + etoposide, or carboplatin + paclitaxel. Ovarian Cancer Combination Therapy

用以治療卵巢癌之治療劑包括5-氟尿嘧啶、白蛋白結合型太平洋紫杉醇、六甲蜜胺、阿那曲唑、貝伐單抗、卡培他濱、卡鉑、順鉑、環磷醯胺、多西紫杉醇、阿黴素、依託泊苷、依西美坦、吉西他濱、異環磷醯胺、伊立替康、來曲唑、乙酸亮丙瑞林、微脂體阿黴素、乙酸甲地孕酮、美法侖、奧拉帕尼、奧沙利鉑、太平洋紫杉醇、帕唑帕尼、培美曲塞、泰莫西芬、拓撲替康、長春瑞濱、及其任何組合。 胰臟癌組合療法 Treatments for ovarian cancer include 5-fluorouracil, albumin-bound paclitaxel, hexamethylmelamine, anastrozole, bevacizumab, capecitabine, carboplatin, cisplatin, cyclophosphamide, docetaxel, doxorubicin, etoposide, exemestane, gemcitabine, isocyclophosphamide, irinotecan, letrozole, leuprolide acetate, liposomal doxorubicin, megestrol acetate, melphalan, olaparib, oxaliplatin, paclitaxel, pazopanib, pemetrexed, tamoxifen, topotecan, vinorelbine, and any combination thereof. Pancreatic cancer combination therapy

用以治療胰臟癌之治療劑包括5-FU、亞葉酸、奧沙利鉑、伊立替康、吉西他濱、白蛋白結合型太平洋紫杉醇(Abraxane ®)、FOLFIRINOX、及其組合。在一些實施例中，用以治療胰臟癌之治療劑包括5-FU +亞葉酸+奧沙利鉑+伊立替康、5-FU +奈米微脂體伊立替康、亞葉酸+奈米微脂體伊立替康、及吉西他濱+白蛋白結合型太平洋紫杉醇。 前列腺癌組合療法 Treatments for pancreatic cancer include 5-FU, leucovorin, oxaliplatin, irinotecan, gemcitabine, albumin-bound paclitaxel ( Abraxane® ), FOLFIRINOX, and combinations thereof. In some practices, treatments for pancreatic cancer include 5-FU + leucovorin + oxaliplatin + irinotecan, 5-FU + nano-liposomal irinotecan, leucovorin + nano-liposomal irinotecan, and gemcitabine + albumin-bound paclitaxel. Combination therapy for prostate cancer.

用以治療前列腺癌之治療劑包括恩雜魯胺(Xtandi ®)、亮丙瑞林、曲氟尿苷、替吡嘧啶（隆瑟夫）、卡巴他賽、潑尼松、阿比特龍(Zytiga ®)、多西紫杉醇、米托蒽醌、比卡魯胺、LHRH、氟他胺、ADT、薩必沙布林(Veru-111)、及其組合。在一些實施例中，用以治療前列腺癌之治療劑包括恩雜魯胺+亮丙瑞林、曲氟尿苷+替吡嘧啶（朗斯弗）、卡巴他賽+潑尼松、阿比特龍+潑尼松、多西紫杉醇+潑尼松、米托蒽醌+潑尼松、比卡魯胺+ LHRH、氟他胺+ LHRH、亮丙瑞林+氟他胺、及阿比特龍+潑尼松+ ADT。 例示性實施例 Treatments for prostate cancer include enzalutamide ( Xtandi® ), leuprorelin, trifluuridine, tepirimidine (Lonzel), cabazitaxel, phenylephrine, abiraterone ( Zytiga® ), docetaxel, mitoxantrone, bicalutamide, LHRH, flutamide, ADT, sabizabulene (Veru-111), and combinations thereof. In some embodiments, treatments for prostate cancer include enzalutamide + leuprorelin, trifluuridine + tepiramycin (Lonsifir), cabazitaxel + nifedipine, abiraterone + nifedipine, docetaxel + nifedipine, mitoxantrone + nifedipine, bicalutamide + LHRH, flutamide + LHRH, leuprorelin + flutamide, and abiraterone + nifedipine + ADT. Exemplary embodiments

實施例1.一種抗FAP抗體或其抗原結合片段，其包含重鏈互補決定區HCDR1、HCDR2、及HCDR3、及輕鏈互補決定區LCDR1、LCDR2、及LCDR3，其中 LCDR1包含如KTNQNVDYX 1GNTFMH (SEQ ID NO: 23)所示之胺基酸序列，其中X 1係N或S，LCDR2包含如LASNLAS (SEQ ID NO: 24)所示之胺基酸序列，LCDR3包含如QQSRNLPYT (SEQ ID NO: 25)所示之胺基酸序列；HCDR1包含如IYGVN (SEQ ID NO: 26)所示之胺基酸序列，HCDR2包含如AIWSGGRKDYX 2LSLKS (SEQ ID NO: 27)所示之胺基酸序列，其中X 2係N或S，且HCDR3包含如SQDMPGYFDY (SEQ ID NO: 28)所示之胺基酸序列；或 LCDR1包含如SASSRVGYMH (SEQ ID NO: 29)所示之胺基酸序列，LCDR2包含如DTSKLAS (SEQ ID NO: 30)所示之胺基酸序列，LCDR3包含如FQGSGYPFT (SEQ ID NO: 31)所示之胺基酸序列；HCDR1包含如TAGMSVG (SEQ ID NO: 32)所示之胺基酸序列，HCDR2包含如DIWWDDKKHYNPSLKD (SEQ ID NO: 33)所示之胺基酸序列，HCDR3包含如DMIFNFYFDV (SEQ ID NO: 34)所示之胺基酸序列。 Example 1. An anti-FAP antibody or its antigen-binding fragment, comprising heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises an amino acid sequence as shown in KTNQNVDYX 1 GNTFMH (SEQ ID NO: 23), where X1 is N or S; LCDR2 comprises an amino acid sequence as shown in LASNLAS (SEQ ID NO: 24); LCDR3 comprises an amino acid sequence as shown in QQSRNLPYT (SEQ ID NO: 25); HCDR1 comprises an amino acid sequence as shown in IYGVN (SEQ ID NO: 26); HCDR2 comprises an amino acid sequence as shown in AIWSGGRKDYX 2 LSLKS (SEQ ID NO: 27), where X1 is N or S; LCDR2 comprises an amino acid sequence as shown in AIWSGGRKDYX 2 LSLKS (SEQ ID NO: 27), where X1 is N or S; LCDR2 comprises an amino acid sequence as shown in LASNLAS (SEQ ID NO: 24); LCDR3 comprises an amino acid sequence as shown in QQSRNLPYT (SEQ ID NO: 25); HCDR1 comprises an amino acid sequence as shown in IYGVN (SEQ ID NO: 26); HCDR2 comprises an amino acid sequence as shown in AIWSGGRKDYX 2 LSLKS (SEQ ID NO: 27); where X1 is N or S; LCDR2 comprises an amino acid sequence as shown in LASNLAS (SEQ ID NO: 24); LCDR3 comprises an amino acid sequence as shown in QQSRNLPYT (SEQ ID NO: 25); LCDR1 comprises an amino acid sequence as shown in IYGVN (SEQ ID NO: 26); LCDR2 comprises an amino acid sequence as shown in AIWSGGRKDYX 2 LSLKS (SEQ ID NO: 27); LCDR3 2 is N or S, and HCDR3 contains an amino acid sequence as shown in SQDMPGYFDY (SEQ ID NO: 28); or LCDR1 contains an amino acid sequence as shown in SASSRVGYMH (SEQ ID NO: 29), LCDR2 contains an amino acid sequence as shown in DTSKLAS (SEQ ID NO: 30), LCDR3 contains an amino acid sequence as shown in FQGSGYPFT (SEQ ID NO: 31); HCDR1 contains an amino acid sequence as shown in TAGMSVG (SEQ ID NO: 32), HCDR2 contains an amino acid sequence as shown in DIWWDDKKHYNPSLKD (SEQ ID NO: 33), and HCDR3 contains an amino acid sequence as shown in DMIFNFYFDV (SEQ ID NO: 34).

實施例2.如實施例1之抗FAP抗體或其抗原結合片段，其包含重鏈可變區(VH)及輕鏈(VL)，其中該VH包括如實施例1之HCDR1、HCDR2、及HCDR3，及/或該VL包括如實施例1之LCDR1、LCDR2、及LCDR3。Example 2. The anti-FAP antibody or its antigen-binding fragment as in Example 1 includes a variable heavy chain region (VH) and a light chain (VL), wherein the VH includes HCDR1, HCDR2 and HCDR3 as in Example 1, and/or the VL includes LCDR1, LCDR2 and LCDR3 as in Example 1.

實施例3.如實施例1至2中任一項之抗FAP抗體或其抗原結合片段，其包含衍生自免疫球蛋白之可結晶片段(Fc)區。Example 3. An anti-FAP antibody or its antigen-binding fragment, as described in any of Examples 1 to 2, includes a crystallizable fragment (Fc) region derived from an immunoglobulin.

實施例4.如實施例3之抗FAP抗體或其抗原結合片段，其中該Fc片段係衍生自IgG1、IgG2、IgG3、或IgG4，可選地，該Fc片段係衍生自IgG4。Example 4. The anti-FAP antibody or its antigen-binding fragment as in Example 3, wherein the Fc fragment is derived from IgG1, IgG2, IgG3 or IgG4, and optionally, the Fc fragment is derived from IgG4.

實施例5.如實施例3或4之抗FAP抗體或其抗原結合片段，其中該Fc片段包含突變S228P。Example 5. An anti-FAP antibody or its antigen-binding fragment, as in Example 3 or 4, wherein the Fc fragment contains the mutant S228P.

實施例6.如實施例5之抗FAP抗體或其抗原結合片段，其包含 (1)  具有SEQ ID NO: 1所述之胺基酸序列的重鏈、及具有SEQ ID NO: 2所述之胺基酸序列的輕鏈；或 (2)  具有SEQ ID NO: 4所述之胺基酸序列的重鏈、及具有SEQ ID NO: 3所述之胺基酸序列的輕鏈。 Example 6. The anti-FAP antibody or its antigen-binding fragment as described in Example 5, comprising: (1) a heavy chain having the amino acid sequence of SEQ ID NO: 1 and a light chain having the amino acid sequence of SEQ ID NO: 2; or (2) a heavy chain having the amino acid sequence of SEQ ID NO: 4 and a light chain having the amino acid sequence of SEQ ID NO: 3.

實施例7.如實施例1至6中任一項之抗FAP抗體或其抗原結合片段，其中該抗FAP抗體或其抗原結合片段與人類、食蟹獼猴、及小鼠FAP交叉反應。Example 7. An anti-FAP antibody or antigen-binding fragment thereof, as described in any of Examples 1 to 6, wherein the anti-FAP antibody or antigen-binding fragment thereof cross-reacts with human, cynomolgus monkey, and mouse FAP.

實施例8.一種雙特異性抗體或其抗原結合片段，其包含FAP抗原結合部份及能夠刺激免疫細胞的細胞介素部份，其中該FAP抗原結合部份包含： LCDR1包含如KTNQNVDYX 1GNTFMH (SEQ ID NO: 23)所示之胺基酸序列，其中X 1係N或S，LCDR2包含如LASNLAS (SEQ ID NO: 24)所示之胺基酸序列，LCDR3包含如QQSRNLPYT (SEQ ID NO: 25)所示之胺基酸序列；HCDR1包含如IYGVN (SEQ ID NO: 26)所示之胺基酸序列，HCDR2包含如AIWSGGRKDYX 2LSLKS (SEQ ID NO: 27)所示之胺基酸序列，其中X 2係N或S，HCDR3包含如SQDMPGYFDY (SEQ ID NO: 28)所示之胺基酸序列；或 LCDR1包含如SASSRVGYMH (SEQ ID NO: 29)所示之胺基酸序列，LCDR2包含如DTSKLAS (SEQ ID NO: 30)所示之胺基酸序列，LCDR3包含如FQGSGYPFT (SEQ ID NO: 31)所示之胺基酸序列；HCDR1包含如TAGMSVG (SEQ ID NO: 32)所示之胺基酸序列，HCDR2包含如DIWWDDKKHYNPSLKD (SEQ ID NO: 33)所示之胺基酸序列，HCDR3包含如DMIFNFYFDV (SEQ ID NO: 34)所示之胺基酸序列。 Example 8. A bispecific antibody or its antigen-binding fragment thereof, comprising an FAP antigen-binding portion and a cytokine portion capable of stimulating immune cells, wherein the FAP antigen-binding portion comprises: LCDR1 comprising an amino acid sequence as shown in KTNQNVDYX 1 GNTFMH (SEQ ID NO: 23), wherein X1 is N or S; LCDR2 comprising an amino acid sequence as shown in LASNLAS (SEQ ID NO: 24); LCDR3 comprising an amino acid sequence as shown in QQSRNLPYT (SEQ ID NO: 25); HCDR1 comprising an amino acid sequence as shown in IYGVN (SEQ ID NO: 26); HCDR2 comprising an amino acid sequence as shown in AIWSGGRKDYX 2 LSLKS (SEQ ID NO: 27), wherein X2 is N or S; HCDR3 comprising an amino acid sequence as shown in SQDMPGYFDY (SEQ ID NO: 27). The amino acid sequence shown in NO: 28); or LCDR1 contains the amino acid sequence shown in SASSRVGYMH (SEQ ID NO: 29), LCDR2 contains the amino acid sequence shown in DTSKLAS (SEQ ID NO: 30), LCDR3 contains the amino acid sequence shown in FQGSGYPFT (SEQ ID NO: 31); HCDR1 contains the amino acid sequence shown in TAGMSVG (SEQ ID NO: 32), HCDR2 contains the amino acid sequence shown in DIWWDDKKHYNPSLKD (SEQ ID NO: 33), and HCDR3 contains the amino acid sequence shown in DMIFNFYFDV (SEQ ID NO: 34).

實施例9.如實施例8之雙特異性抗體或其抗原結合片段，其中該FAP抗原結合部份包含與VL配對的至少一個VH，其中該VH包括如實施例8之HCDR1、HCDR2、及HCDR3，及/或該VL包括如實施例8之LCDR1、LCDR2、及LCDR3。Example 9. A bispecific antibody or antigen-binding fragment thereof as in Example 8, wherein the FAP antigen-binding portion includes at least one VH paired with VL, wherein the VH includes HCDR1, HCDR2, and HCDR3 as in Example 8, and/or the VL includes LCDR1, LCDR2, and LCDR3 as in Example 8.

實施例10.如實施例8或9之雙特異性抗體或其抗原結合片段，其在該FAP抗原結合部份之N端包含衍生自免疫球蛋白之Fc片段。Example 10. The bispecific antibody or its antigen-binding fragment, as in Example 8 or 9, contains an Fc fragment derived from immunoglobulin at the N-terminus of the FAP antigen-binding region.

實施例11.如實施例10之雙特異性抗體或其抗原結合片段，其中該Fc片段係衍生自IgG1、IgG2、IgG3、或IgG4，可選地，該Fc片段係衍生自IgG4。Example 11. The bispecific antibody or its antigen-binding fragment as in Example 10, wherein the Fc fragment is derived from IgG1, IgG2, IgG3 or IgG4, and optionally, the Fc fragment is derived from IgG4.

實施例12.如實施例10或11之雙特異性抗體或其抗原結合片段，其中該Fc片段包含突變S228P。Example 12. A bispecific antibody or antigen-binding fragment thereof, such as in Example 10 or 11, wherein the Fc fragment contains the mutant S228P.

實施例13.如實施例8至12中任一項之雙特異性抗體或其抗原結合片段，其中該Fc片段包含選自由下列所組成之群組的一或多個修飾：鈕扣、DDKK、CH3之靜電轉向、DuoBody、SEEDbodies、cFAE、XmAb、Azymetric、及BEAT ®，可選地，該Fc片段包含修飾鈕扣及/或DDKK。 Example 13. A bispecific antibody or antigen-binding fragment thereof of any of Examples 8 to 12, wherein the Fc fragment comprises one or more modifications selected from the group consisting of: button, DDKK, electrostatic switching of CH3, DuoBody, SEEDbodies, cFAE, XmAb, Azymetric, and BEAT® , optionally, the Fc fragment comprises a button and/or DDKK modification.

實施例14.如實施例8至13中任一項之雙特異性抗體或其抗原結合片段，其中該細胞介素部份係在該雙特異性抗體或其抗原結合片段之C端。Example 14. A bispecific antibody or antigen-binding fragment thereof, as described in any of Examples 8 to 13, wherein the interleukin portion is located at the C-terminus of the bispecific antibody or antigen-binding fragment thereof.

實施例15.如實施例8至14中任一項之雙特異性抗體或其抗原結合片段，其中該細胞介素部份係可操作地連接至該Fc片段之該C端。Example 15. A bispecific antibody or antigen-binding fragment thereof, as described in any of Examples 8 to 14, wherein the interleukin portion is operatively linked to the C-terminus of the Fc fragment.

實施例16.如實施例8至15中任一項之雙特異性抗體或其抗原結合片段，其中該細胞介素部份經由連接子連接至該Fc片段。Example 16. A bispecific antibody or antigen-binding fragment thereof, as described in any of Examples 8 to 15, wherein the interleukin portion is linked to the Fc fragment via a linker.

實施例17.如實施例8至16中任一項之雙特異性抗體或其抗原結合片段，其中該細胞介素部份包含第一細胞介素部份及第二細胞介素部份，該第一細胞介素部份包含一個LIGHT突變體且該第二細胞介素部份包含串聯連接的兩個LIGHT突變體，該LIGHT突變體包括SEQ ID NO: 17或18所述之胺基酸序列。Example 17. A bispecific antibody or antigen-binding fragment thereof as described in any of Examples 8 to 16, wherein the interleukin portion comprises a first interleukin portion and a second interleukin portion, the first interleukin portion comprising a LIGHT mutant and the second interleukin portion comprising two LIGHT mutants connected in tandem, the LIGHT mutant comprising the amino acid sequence described in SEQ ID NO: 17 or 18.

實施例18.如實施例8至17中任一項之雙特異性抗體或其抗原結合片段，其包含第一重鏈、第二重鏈、及分別與該第一重鏈及該第二重鏈配對之兩條輕鏈，其中 (1)  該第一重鏈包含與SEQ ID NO: 5所述之胺基酸序列具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%同一性之胺基酸序列；該第二重鏈包含與SEQ ID NO: 6所述之胺基酸序列具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%同一性之胺基酸序列；該輕鏈包含與SEQ ID NO: 3所述之胺基酸序列具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%同一性之胺基酸序列； (2)  該第一重鏈包含與SEQ ID NO: 8所述之胺基酸序列具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%同一性之胺基酸序列；該第二重鏈包含與SEQ ID NO: 9所述之胺基酸序列具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%同一性之胺基酸序列；該輕鏈包含與SEQ ID NO: 7所述之胺基酸序列具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%同一性之胺基酸序列； (3)  該第一重鏈包含與SEQ ID NO: 10所述之胺基酸序列具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%同一性之胺基酸序列；該第二重鏈包含與SEQ ID NO: 11所述之胺基酸序列具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%同一性之胺基酸序列；該輕鏈包含與SEQ ID NO: 3所述之胺基酸序列具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%同一性之胺基酸序列。 Example 18. A bispecific antibody or antigen-binding fragment thereof of any one of Examples 8 to 17, comprising a first heavy chain, a second heavy chain, and two light chains respectively paired with the first heavy chain and the second heavy chain, wherein (1) The first heavy chain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with the amino acid sequence described in SEQ ID NO: 5; the second heavy chain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with the amino acid sequence described in SEQ ID NO: 6; the light chain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with the amino acid sequence described in SEQ ID NO: 6; (3) The amino acid sequence described herein has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity with the amino acid sequence described in SEQ ID NO: 8; (2) The first heavy chain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity with the amino acid sequence described in SEQ ID NO: 9; the second heavy chain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity with the amino acid sequence described in SEQ ID NO: 9; the light chain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity with the amino acid sequence described in SEQ ID NO: 9; 7. The amino acid sequence described herein has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity with the amino acid sequence described in SEQ ID NO: 10; (3) The first heavy chain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity with the amino acid sequence described in SEQ ID NO: 10; the second heavy chain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity with the amino acid sequence described in SEQ ID NO: 11; the light chain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity with the amino acid sequence described in SEQ ID NO: 11; The amino acid sequence described in 3 has an amino acid sequence identity of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

實施例19.如實施例8至18中任一項之雙特異性抗體或其抗原結合片段，其包含下列： (1)  具有SEQ ID NO: 5所述之基酸序列的該第一重鏈、具有SEQ ID NO: 6所述之胺基酸序列的該第二重鏈、及具有SEQ ID NO: 3所述之胺基酸序列的該輕鏈； (2)  具有SEQ ID NO: 8所述之胺基酸序列的該第一重鏈、具有SEQ ID NO: 9所述之胺基酸序列的該第二重鏈、及具有SEQ ID NO: 7所述之胺基酸序列的該輕鏈； (3)  具有SEQ ID NO: 10所述之胺基酸序列的該第一重鏈、具有SEQ ID NO: 11所述之胺基酸序列的該第二重鏈、及具有SEQ ID NO: 3所述之胺基酸序列的該輕鏈。 Example 19. A bispecific antibody or antigen-binding fragment thereof of any one of Examples 8 to 18, comprising the following: (1) the first heavy chain having the amino acid sequence of SEQ ID NO: 5, the second heavy chain having the amino acid sequence of SEQ ID NO: 6, and the light chain having the amino acid sequence of SEQ ID NO: 3; (2) the first heavy chain having the amino acid sequence of SEQ ID NO: 8, the second heavy chain having the amino acid sequence of SEQ ID NO: 9, and the light chain having the amino acid sequence of SEQ ID NO: 7; (3) the first heavy chain having the amino acid sequence of SEQ ID NO: 10, the second heavy chain having the amino acid sequence of SEQ ID NO: 11, and the light chain having the amino acid sequence of SEQ ID NO: 12. The light chain of the amino acid sequence described in section 3.

實施例20.如實施例8至19中任一項之雙特異性抗體或其抗原結合片段，其中該雙特異性抗體或其抗原結合片段以不大於20 nM、15 nM、10 nM、或5 nM之解離常數(KD)結合至FAP。Example 20. A bispecific antibody or antigen-binding fragment thereof, as described in any of Examples 8 to 19, wherein the bispecific antibody or antigen-binding fragment thereof binds to the FAP with a dissociation constant (KD) not greater than 20 nM, 15 nM, 10 nM, or 5 nM.

實施例21.如實施例8至20中任一項之雙特異性抗體或其抗原結合片段，其中該雙特異性抗體或其抗原結合片段以不大於20 nM、15 nM、10 nM、或5 nM之解離常數(KD)結合至LTßR。Example 21. A bispecific antibody or antigen-binding fragment thereof, as described in any of Examples 8 to 20, wherein the bispecific antibody or antigen-binding fragment thereof binds to LTßR with a dissociation constant (KD) not greater than 20 nM, 15 nM, 10 nM, or 5 nM.

實施例22.如實施例8至21中任一項之雙特異性抗體或其抗原結合片段，其中該雙特異性抗體或其抗原結合片段幾乎不結合至人類或食蟹獼猴HVEM。Example 22. A bispecific antibody or antigen-binding fragment thereof, as described in any of Examples 8 to 21, wherein the bispecific antibody or antigen-binding fragment thereof binds substantially not to human or cynomolgus monkey HVEM.

實施例23.如實施例8至22中任一項之雙特異性抗體或其抗原結合片段，其中雙特異性抗體或其抗原結合片段中之該LIGHT突變體能夠降低與DcR3之結合親和力。Example 23. A bispecific antibody or antigen-binding fragment thereof, as described in any of Examples 8 to 22, wherein the LIGHT mutant in the bispecific antibody or antigen-binding fragment thereof is capable of reducing the binding affinity to DcR3.

實施例24.如實施例8至23中任一項之雙特異性抗體或其抗原結合片段，其中該雙特異性抗體或其抗原結合片段特異性結合至人類FAP，且/或不結合至DPPIV。Example 24. A bispecific antibody or antigen-binding fragment thereof, as described in any of Examples 8 to 23, wherein the bispecific antibody or antigen-binding fragment thereof specifically binds to human FAP and/or does not bind to DPPIV.

實施例25.一種經單離多核苷酸，其編碼如實施例1至7中任一項之抗FAP抗體或其抗原結合片段或如實施例8至24中任一項之雙特異性抗體或其抗原結合片段的任何序列。Example 25. A single isolated polynucleotide encoding any sequence of an anti-FAP antibody or its antigen-binding fragment as in any of Examples 1 to 7, or a bispecific antibody or its antigen-binding fragment as in any of Examples 8 to 24.

實施例26.一種構築體，其包含如實施例25之多核苷酸。Example 26. A structure comprising a polynucleotide as in Example 25.

實施例27.一種抗體表現系統，其包含包括如實施例25之經單離多核苷酸或具有整合有如實施例25之外源多核苷酸的基因體的該構築體，其中較佳地，該表現系統係細胞表現系統。Example 27. An antibody expression system comprising the construct including a single polynucleotide as in Example 25 or having a genotype integrating an exogenous polynucleotide as in Example 25, wherein preferably, the expression system is a cell expression system.

實施例28.一種用於生產如實施例1至7中任一項之抗FAP抗體或其抗原結合片段或如實施例8至24中任一項之雙特異性抗體或其抗原結合片段的方法，其包含：在適合表現該抗體的條件下，使用如實施例27之抗體表現系統表現該抗體或融合蛋白。Example 28. A method for producing an anti-FAP antibody or an antigen-binding fragment thereof as described in any one of Examples 1 to 7, or a bispecific antibody or an antigen-binding fragment thereof as described in any one of Examples 8 to 24, comprising: expressing the antibody or fusion protein using an antibody expression system as described in Example 27 under conditions suitable for expressing the antibody.

實施例29.一種醫藥組成物，其包含如實施例1至7中任一項之抗FAP抗體或其抗原結合片段或如實施例8至24中任一項之雙特異性抗體或其抗原結合片段、及醫藥上可接受之載劑。Example 29. A pharmaceutical composition comprising an anti-FAP antibody or an antigen-binding fragment thereof as in any of Examples 1 to 7, or a bispecific antibody or an antigen-binding fragment thereof as in any of Examples 8 to 24, and a pharmaceutically acceptable delivery vehicle.

實施例30.一種套組，其包含如實施例1至7中任一項之抗FAP抗體或其抗原結合片段、如實施例8至24中任一項之雙特異性抗體或其抗原結合片段、如實施例25之經單離多核苷酸、或如實施例26之構築體。Example 30. A kit comprising an anti-FAP antibody or an antigen-binding fragment thereof as in any of Examples 1 to 7, a bispecific antibody or an antigen-binding fragment thereof as in any of Examples 8 to 24, a monoisopropyl nucleotide as in Example 25, or a building block as in Example 26.

實施例31.一種如實施例1至7中任一項之抗FAP抗體或其抗原結合片段、如實施例8至24中任一項之雙特異性抗體或其抗原結合片段、或如實施例29之醫藥組成物於製造用於預防、診斷、或治療疾病、病症、或病況之治療劑的用途。Example 31. Use of an anti-FAP antibody or antigen-binding fragment thereof as described in any of Examples 1 to 7, a bispecific antibody or antigen-binding fragment thereof as described in any of Examples 8 to 24, or a pharmaceutical composition as described in Example 29 in the manufacture of a treatment for the prevention, diagnosis, or treatment of a disease, condition, or illness.

實施例32.如實施例31之用途，其中該疾病、病症、或病況包含腫瘤疾病。Example 32. As in Example 31, wherein the disease, symptom, or condition includes a tumor.

實施例33.如實施例31或32之用途，其中至少一個腫瘤細胞表現FAP。Example 33. As used in Examples 31 or 32, in which at least one tumor cell exhibits FAP.

實施例34.如實施例31至33中任一項之用途，其中該等腫瘤疾病係實體腫瘤。Example 34. As in any of Examples 31 to 33, wherein the tumor disease is a solid tumor.

實施例35.如實施例31至34中任一項之用途，其中該腫瘤包含胃癌、肝癌、肺癌、結腸直腸癌、乳癌、前列腺癌、皮膚癌、骨癌、多發性骨髓瘤、神經膠質瘤、卵巢癌、胰臟癌、子宮頸癌、甲狀腺癌、喉癌、急性骨髓性白血病、慢性骨髓性白血病、急性淋巴母細胞白血病、慢性淋巴母細胞白血病、腦腫瘤、神經母細胞瘤、視網膜母細胞瘤、頭頸癌、唾液腺癌、及淋巴瘤。Example 35. As used in any of Examples 31 to 34, wherein the tumor includes gastric cancer, liver cancer, lung cancer, colorectal cancer, breast cancer, prostate cancer, skin cancer, bone cancer, multiple myeloma, glioma, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and lymphoma.

實施例36.一種用於治療有需要之對象的方法，其包含向該對象投予治療有效量的如實施例1至7中任一項之抗FAP抗體或其抗原結合片段、如實施例8至24中任一項之雙特異性抗體或其抗原結合片段、或如實施例29之醫藥組成物。Example 36. A method for treating a subject in need, comprising administering to the subject a therapeutically effective amount of an anti-FAP antibody or an antigen-binding fragment thereof, such as any one of Examples 1 to 7, a bispecific antibody or an antigen-binding fragment thereof, such as any one of Examples 8 to 24, or a pharmaceutical composition such as Example 29.

實施例37.一種用於降低腫瘤生長速率或腫瘤細胞數目的方法，其包含使腫瘤細胞與有效量的如實施例1至7中任一項之抗FAP抗體或其抗原結合片段、如實施例8至24中任一項之雙特異性抗體或其抗原結合片段、或如實施例29之醫藥組成物接觸。Example 37. A method for reducing tumor growth rate or number of tumor cells, comprising contacting tumor cells with an effective amount of an anti-FAP antibody or antigen-binding fragment thereof, as in any of Examples 1 to 7, a bispecific antibody or antigen-binding fragment thereof, as in any of Examples 8 to 24, or a pharmaceutical composition, as in Example 29.

實施例38.一種殺滅腫瘤細胞之方法，其包含使腫瘤細胞與有效量的如實施例1至7中任一項之抗FAP抗體或其抗原結合片段、如實施例8至24中任一項之雙特異性抗體或其抗原結合片段、或如實施例29之醫藥組成物接觸。Example 38. A method for killing tumor cells, comprising contacting the tumor cells with an effective amount of an anti-FAP antibody or its antigen-binding fragment as in any of Examples 1 to 7, a bispecific antibody or its antigen-binding fragment as in any of Examples 8 to 24, or a pharmaceutical composition as in Example 29.

實施例39.一種抗FAP抗體或其抗原結合片段，其包含包括重鏈互補決定區HCDR1、HCDR2、及HCDR3的重鏈可變區(heavy chain variable region, VH)、及包括輕鏈互補決定區LCDR1、LCDR2、及LCDR3的輕鏈可變區(light chain variable region, VL)，其中 LCDR1包含如KTNQNVDYX 1GNTFMH (SEQ ID NO: 23)所示之胺基酸序列，其中X 1係N或S，LCDR2包含如LASNLAS (SEQ ID NO: 24)所示之胺基酸序列，LCDR3包含如QQSRNLPYT (SEQ ID NO: 25)所示之胺基酸序列，HCDR1包含如IYGVN (SEQ ID NO: 26)所示之胺基酸序列，HCDR2包含如AIWSGGRKDYX 2LSLKS (SEQ ID NO: 27)所示之胺基酸序列，其中X 2係N或S，HCDR3包含如SQDMPGYFDY (SEQ ID NO: 28)所示之胺基酸序列；或 LCDR1包含如SASSRVGYMH (SEQ ID NO: 29)所示之胺基酸序列，LCDR2包含如DTSKLAS (SEQ ID NO: 30)所示之胺基酸序列，LCDR3包含如FQGSGYPFT (SEQ ID NO: 31)所示之胺基酸序列，HCDR1包含如TAGMSVG (SEQ ID NO: 32)所示之胺基酸序列，HCDR2包含如DIWWDDKKHYNPSLKD (SEQ ID NO: 33)所示之胺基酸序列，HCDR3包含如DMIFNFYFDV (SEQ ID NO: 34)所示之胺基酸序列。 Example 39. An anti-FAP antibody or its antigen-binding fragment, comprising a heavy chain variable region (VH) including heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and a light chain variable region (VL) including light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises an amino acid sequence as shown in KTNQNVDYX 1 GNTFMH (SEQ ID NO: 23), where X1 is N or S; LCDR2 comprises an amino acid sequence as shown in LASNLAS (SEQ ID NO: 24); LCDR3 comprises an amino acid sequence as shown in QQSRNLPYT (SEQ ID NO: 25); HCDR1 comprises an amino acid sequence as shown in IYGVN (SEQ ID NO: 26); and HCDR2 comprises an amino acid sequence as shown in AIWSGGRKDYX 2 The amino acid sequence shown in LSLKS (SEQ ID NO: 27), wherein X2 is N or S, HCDR3 contains the amino acid sequence shown in SQDMPGYFDY (SEQ ID NO: 28); or LCDR1 contains the amino acid sequence shown in SASSRVGYMH (SEQ ID NO: 29), LCDR2 contains the amino acid sequence shown in DTSKLAS (SEQ ID NO: 30), LCDR3 contains the amino acid sequence shown in FQGSGYPFT (SEQ ID NO: 31), HCDR1 contains the amino acid sequence shown in TAGMSVG (SEQ ID NO: 32), HCDR2 contains the amino acid sequence shown in DIWWDDKKHYNPSLKD (SEQ ID NO: 33), and HCDR3 contains the amino acid sequence shown in DMIFNFYFDV (SEQ ID NO: 34).

實施例40.如實施例39之抗FAP抗體或其抗原結合片段，其包含 (1)  與胺基酸序列(QVQLKESGPGMVQPSRTLSLTCTVSGFSLSIYGVNWVRQPPGKGLEWIAAIWSGGRKDYNLSLKSRLIISGDTSKSQVLLTMNSLQSEDTAMYFCARSQDMPGYFDYWGQGVMVTVSS)具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、100%同一性的VH、及與胺基酸序列(DIVLTQSPALAVSLGQRATISCKTNQNVDYNGNTFMHWYQQKPGQQPKLLLYLASNLASGIPDRFSGRGSGTDFTLTIDPVEADDTATYYCQQSRNLPYTFGAGTKLEIK)具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、100%同一性的VL；或 (2)  與胺基酸序列(EVQLQESGPGLVKPSETLSLTCTVSGFSLSIYGVNWVRQPPGKGLEWIAAIWSGGRKDYSLSLKSRLTISGDTSKNQVSLKLSSVTAADTAVYYCARSQDMPGYFDYWGQGTLVTVSS)具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、100%同一性的VH、及與胺基酸序列(SIVLTQPPSVSVAPGQTARITCKTNQNVDYSGNTFMHWYQQKPGQQPVLLLYLASNLASGIPERFSGSGSGTTFTLTISRVEAGDEADYYCQQSRNLPYTFGTGTKVTVL)具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、100%同一性的VL。 Example 40. The anti-FAP antibody or its antigen-binding fragment, as in Example 39, comprises (1) having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence (QVQLKESGPGMVQPSRTLSLTCTVSGFSLSIYGVNWVRQPPGKGLEWIAAIWSGGRKDYNLSLKSRLIISGDTSKSQVLLTMNSLQSEDTAMYFCARSQDMPGYFDYWGQGVMVTVSS). VH, and VL having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence (DIVLTQSPALAVSLGQRATISCKTNQNVDYNGNTFMHWYQQKPGQQPKLLLYLASNLASGIPDRFSGRGSGTDFTLTIDPVEADDTATYYCQQSRNLPYTFGAGTKLEIK); or (2) It has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% identity with the amino acid sequence (EVQLQESGPGLVKPSETLSLTCTVSGFSLSIYGVNWVRQPPGKGLEWIAAIWSGGRKDYSLSLKSRLTISGDTSKNQVSLKLSSVTAADTAVYYCARSQDMPGYFDYWGQGTLVTVSS). The VH sequence and the VL sequence (SIVLTQPPSVSVAPGQTARITCKTNQNVDYSGNTFMHWYQQKPGQQPVLLLYLASNLASGIPERFSGSGSGTTFTLTISRVEAGDEADYYCQQSRNLPYTFGTGTKVTVL) have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% identity with the amino acid sequence (SIVLTQPPSVSVAPGQTARITCKTNQNVDYSGNTFMHWYQQKPGQQPVLLLYLASNLASGIPERFSGSGSGTTFTLTISRVEAGDEADYYCQQSRNLPYTFGTGTKVTVL) respectively.

實施例41.如實施例39或40之抗FAP抗體或其抗原結合片段，其包含衍生自免疫球蛋白之可結晶片段(Fc)區。Example 41. The anti-FAP antibody or its antigen-binding fragment, such as in Example 39 or 40, includes a crystallizable fragment (Fc) region derived from an immunoglobulin.

實施例42.如實施例41之抗FAP抗體或其抗原結合片段，其中該Fc片段係衍生自IgG1、IgG2、IgG3、或IgG4，可選地，該Fc片段係衍生自IgG4。Example 42. The anti-FAP antibody or its antigen-binding fragment as in Example 41, wherein the Fc fragment is derived from IgG1, IgG2, IgG3 or IgG4, and optionally, the Fc fragment is derived from IgG4.

實施例43.如實施例41或42之抗FAP抗體或其抗原結合片段，其中該Fc片段包含突變S228P或LALAPG突變。Example 43. An anti-FAP antibody or its antigen-binding fragment, such as in Example 41 or 42, wherein the Fc fragment contains the S228P or LALAPG mutation.

實施例44.如實施例43之抗FAP抗體或其抗原結合片段，其包含 (1)  具有SEQ ID NO: 1所述之胺基酸序列的重鏈、及具有SEQ ID NO: 2所述之胺基酸序列的輕鏈；或 (2)  具有SEQ ID NO: 3所述之胺基酸序列的重鏈、及具有SEQ ID NO: 4所述之胺基酸序列的輕鏈。 Example 44. The anti-FAP antibody or its antigen-binding fragment as described in Example 43, comprising (1) a heavy chain having the amino acid sequence of SEQ ID NO: 1 and a light chain having the amino acid sequence of SEQ ID NO: 2; or (2) a heavy chain having the amino acid sequence of SEQ ID NO: 3 and a light chain having the amino acid sequence of SEQ ID NO: 4.

實施例45.如實施例39至44中任一項之抗FAP抗體或其抗原結合片段，其中該抗FAP抗體或其抗原結合片段與人類、食蟹獼猴、及小鼠FAP交叉反應Example 45. An anti-FAP antibody or antigen-binding fragment thereof, as described in any of Examples 39 to 44, wherein the anti-FAP antibody or antigen-binding fragment thereof cross-reacts with human, cynomolgus monkey, and mouse FAP.

實施例46.融合蛋白，其包含FAP抗原結合部份、及能夠刺激免疫細胞的細胞介素部份，其中該FAP抗原結合部份包含包括重鏈互補決定區HCDR1、HCDR2、及HCDR3的重鏈可變區(VH)、及包括輕鏈互補決定區LCDR1、LCDR2、及LCDR3的輕鏈可變區(VL)，其中 LCDR1包含如KTNQNVDYX 1GNTFMH (SEQ ID NO: 23)所示之胺基酸序列，其中X 1係N或S，LCDR2包含如LASNLAS (SEQ ID NO: 24)所示之胺基酸序列，LCDR3包含如QQSRNLPYT (SEQ ID NO: 25)所示之胺基酸序列；HCDR1包含如IYGVN (SEQ ID NO: 26)所示之胺基酸序列，HCDR2包含如AIWSGGRKDYX 2LSLKS (SEQ ID NO: 27)所示之胺基酸序列，其中X 2係N或S，HCDR3包含如SQDMPGYFDY (SEQ ID NO: 28)所示之胺基酸序列；或 LCDR1包含如SASSRVGYMH (SEQ ID NO: 29)所示之胺基酸序列，LCDR2包含如DTSKLAS (SEQ ID NO: 30)所示之胺基酸序列，LCDR3包含如FQGSGYPFT (SEQ ID NO: 31)所示之胺基酸序列；HCDR1包含如TAGMSVG (SEQ ID NO: 32)所示之胺基酸序列，HCDR2包含如DIWWDDKKHYNPSLKD (SEQ ID NO: 33)所示之胺基酸序列，HCDR3包含如DMIFNFYFDV (SEQ ID NO: 34)所示之胺基酸序列。 Example 46. A fusion protein comprising an FAP antigen-binding region and a cytokine region capable of stimulating immune cells, wherein the FAP antigen-binding region comprises a heavy chain variable region (VH) including heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and a light chain variable region (VL) including light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises an amino acid sequence as shown in KTNQNVDYX 1 GNTFMH (SEQ ID NO: 23), where X1 is N or S; LCDR2 comprises an amino acid sequence as shown in LASNLAS (SEQ ID NO: 24); LCDR3 comprises an amino acid sequence as shown in QQSRNLPYT (SEQ ID NO: 25); HCDR1 comprises an amino acid sequence as shown in IYGVN (SEQ ID NO: 25). 26) The amino acid sequence shown, HCDR2 contains the amino acid sequence shown as AIWSGGRKDYX 2 LSLKS (SEQ ID NO: 27), where X 2 is N or S, HCDR3 contains the amino acid sequence shown as SQDMPGYFDY (SEQ ID NO: 28); or LCDR1 contains the amino acid sequence shown as SASSRVGYMH (SEQ ID NO: 29), LCDR2 contains the amino acid sequence shown as DTSKLAS (SEQ ID NO: 30), LCDR3 contains the amino acid sequence shown as FQGSGYPFT (SEQ ID NO: 31); HCDR1 contains the amino acid sequence shown as TAGMSVG (SEQ ID NO: 32), HCDR2 contains the amino acid sequence shown as DIWWDDKKHYNPSLKD (SEQ ID NO: 33), HCDR3 contains the amino acid sequence shown as DMIFNFYFDV (SEQ ID NO: 34).

實施例47.如實施例46之融合蛋白，其中該FAP抗原結合部份包含 (1)  與胺基酸序列(QVQLKESGPGMVQPSRTLSLTCTVSGFSLSIYGVNWVRQPPGKGLEWIAAIWSGGRKDYNLSLKSRLIISGDTSKSQVLLTMNSLQSEDTAMYFCARSQDMPGYFDYWGQGVMVTVSS)具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、100%同一性的VH、及與胺基酸序列(DIVLTQSPALAVSLGQRATISCKTNQNVDYNGNTFMHWYQQKPGQQPKLLLYLASNLASGIPDRFSGRGSGTDFTLTIDPVEADDTATYYCQQSRNLPYTFGAGTKLEIK)具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、100%同一性的VL；或 (2)  與胺基酸序列(EVQLQESGPGLVKPSETLSLTCTVSGFSLSIYGVNWVRQPPGKGLEWIAAIWSGGRKDYSLSLKSRLTISGDTSKNQVSLKLSSVTAADTAVYYCARSQDMPGYFDYWGQGTLVTVSS)具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、100%同一性的VH、及與胺基酸序列(SIVLTQPPSVSVAPGQTARITCKTNQNVDYSGNTFMHWYQQKPGQQPVLLLYLASNLASGIPERFSGSGSGTTFTLTISRVEAGDEADYYCQQSRNLPYTFGTGTKVTVL)具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、100%同一性的VL。 Example 47. The fusion protein of Example 46, wherein the FAP antigen-binding portion comprises (1) having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence (QVQLKESGPGMVQPSRTLSLTCTVSGFSLSIYGVNWVRQPPGKGLEWIAAIWSGGRKDYNLSLKSRLIISGDTSKSQVLLTMNSLQSEDTAMYFCARSQDMPGYFDYWGQGVMVTVSS) VH, and VL having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence (DIVLTQSPALAVSLGQRATISCKTNQNVDYNGNTFMHWYQQKPGQQPKLLLYLASNLASGIPDRFSGRGSGTDFTLTIDPVEADDTATYYCQQSRNLPYTFGAGTKLEIK); or (2) It has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% identity with the amino acid sequence (EVQLQESGPGLVKPSETLSLTCTVSGFSLSIYGVNWVRQPPGKGLEWIAAIWSGGRKDYSLSLKSRLTISGDTSKNQVSLKLSSVTAADTAVYYCARSQDMPGYFDYWGQGTLVTVSS). The VH sequence and the VL sequence (SIVLTQPPSVSVAPGQTARITCKTNQNVDYSGNTFMHWYQQKPGQQPVLLLYLASNLASGIPERFSGSGSGTTFTLTISRVEAGDEADYYCQQSRNLPYTFGTGTKVTVL) have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% identity with the amino acid sequence (SIVLTQPPSVSVAPGQTARITCKTNQNVDYSGNTFMHWYQQKPGQQPVLLLYLASNLASGIPERFSGSGSGTTFTLTISRVEAGDEADYYCQQSRNLPYTFGTGTKVTVL) respectively.

實施例48.如實施例46或47之融合蛋白，其在該FAP抗原結合部份之N端包含衍生自免疫球蛋白之Fc片段。Example 48. The fusion protein of Example 46 or 47 includes an Fc fragment derived from immunoglobulin at the N-terminus of the FAP antigen-binding region.

實施例49.如實施例48之融合蛋白，其中該Fc片段係衍生自IgG1、IgG2、IgG3、或IgG4，可選地，該Fc片段係衍生自IgG4。Example 49. The fusion protein of Example 48, wherein the Fc fragment is derived from IgG1, IgG2, IgG3 or IgG4, and optionally, the Fc fragment is derived from IgG4.

實施例50.如實施例48或49之融合蛋白，其中該Fc片段包含突變S228P或LALAPG突變。Example 50. A fusion protein such as in Example 48 or 49, wherein the Fc fragment contains the S228P or LALAPG mutation.

實施例51.如實施例46至50中任一項之融合蛋白，其中該Fc片段包含選自由下列所組成之群組的一或多個修飾：鈕扣、DDKK、CH3之靜電轉向、DuoBody、SEEDbodies、cFAE、XmAb、Azymetric、及BEAT ®，可選地，該Fc片段包含修飾鈕扣及/或DDKK。 Example 51. A fusion protein of any of Examples 46 to 50, wherein the Fc fragment comprises one or more modifications selected from the group consisting of: button, DDKK, electrostatic switching of CH3, DuoBody, SEEDbodies, cFAE, XmAb, Azymetric, and BEAT® , optionally, the Fc fragment comprises a button and/or DDKK modification.

實施例52.如實施例46至51中任一項之融合蛋白，其中該細胞介素部份係在該融合蛋白之C端；可選地，該細胞介素部份係可操作地連接至該Fc片段之該C端；可選地，該細胞介素部份經由連接子連接至該Fc片段Example 52. A fusion protein as described in any of Examples 46 to 51, wherein the interleukin portion is located at the C-terminus of the fusion protein; alternatively, the interleukin portion is operatively linked to the C-terminus of the Fc fragment; alternatively, the interleukin portion is linked to the Fc fragment via a linker.

實施例53.如實施例46至52中任一項之融合蛋白，其中該細胞介素部份包含第一細胞介素部份及第二細胞介素部份，該第一細胞介素部份包含一個細胞介素突變體且該第二細胞介素部份包含串聯連接的兩個細胞介素突變體，該第一細胞介素部份及該第二細胞介素部份連接至該Fc片段內之該等蛋白質片段的不同C端。Example 53. A fusion protein as described in any of Examples 46 to 52, wherein the interleukin portion comprises a first interleukin portion and a second interleukin portion, the first interleukin portion comprising an interleukin mutant and the second interleukin portion comprising two interleukin mutants connected in series, the first interleukin portion and the second interleukin portion being linked to different C-termini of the protein fragments within the Fc fragment.

實施例54.如實施例53之融合蛋白，其中該第一細胞介素部份連接至具有孔突變之該Fc片段之該C端，且該第二細胞介素部份連接至具有鈕突變之該Fc片段之該C端Example 54. The fusion protein of Example 53, wherein the first intercytokine portion is linked to the C-terminus of the Fc fragment with a pore mutation, and the second intercytokine portion is linked to the C-terminus of the Fc fragment with a button mutation.

實施例55.如實施例53或54之融合蛋白，其中該細胞介素突變體係LIGHT突變體，該LIGHT突變體包括SEQ ID NO: 17或18所述之胺基酸序列；或 該第一細胞介素部份包含一個淋巴毒素-β突變體，且該第二細胞介素部份包含串聯連接的淋巴毒素-αβ突變體，該淋巴毒素-β突變體包括胺基酸序列(LSPGLPAAHLIGAPLKGQGLGWETTKEQAFLTSGTQFSDAEGLALPQDGLYYLYCLVGYRGRAPPGGGDPQGRSVTLRSSLYRAGGAYGPGTPELLLEGAETVTPVLDPARRQGYGPLWYTSVGFGGLVQLRRGERVYVNISHPDMVDFARGKTFFGAVMVG)，該淋巴毒素-α突變體包括胺基酸序列(AHSTLKPAAHLIGDPSKQNSLLWRANTDRAFLQDGFSLSNNSLLVPTSGIYFVYSQVVFSGKAYSPKATSSPLYLAHEVQLFSSQYPFHVPLLSSQKMVYPGLQEPWLHSMYHGAAFQLTQGDQLSTHTDGIPHLVLSPSTVFFGAFAL)。 Example 55. A fusion protein as in Example 53 or 54, wherein the interleukin mutant is a LIGHT mutant, the LIGHT mutant comprising the amino acid sequence described in SEQ ID NO: 17 or 18; or the first interleukin portion comprises a lymphotoxin-β mutant, and the second interleukin portion comprises tandemly linked lymphotoxin-αβ mutants, the lymphotoxin-β mutant comprising the amino acid sequence (LSPGLPAAHLIGAPLKGQGLGWETTKEQAFLTSGTQFSDAEGLALPQDGLYYLYCLVGYRGRAPPGGGDPQGRSVTLRSSLYRAGGAYGPGTPELLLEGAETVTPVLDPARRQGYGPLWYTSVGFGGLVQLR) RGERVYVNISHPDMVDFARGKTFFGAVMVG), this lymphotoxin-α mutant includes the amino acid sequence (AHSTLKPAAHLIGDPSKQNSLLWRANTDRAFLQDGFSLSNNSLLVPTSGIYFVYSQVVFSGKAYSPKATSSPLYLAHEVQLFSSQYPFHVPLLSSQKMVYPGLQEPWLHSMYHGAAFQLTQGDQLSTHTDGIPHLVLSPSTVFFGAFAL).

實施例56.如實施例46至55中任一項之融合蛋白，其包含第一重鏈、第二重鏈、及分別與該第一重鏈及該第二重鏈配對之兩條輕鏈，其中 (1)  該第一重鏈包含與SEQ ID NO: 3所述之胺基酸序列具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、100%同一性之胺基酸序列；該第二重鏈包含與SEQ ID NO: 5所述之胺基酸序列具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、100%同一性之胺基酸序列；該輕鏈包含與SEQ ID NO: 6所述之胺基酸序列具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、100%同一性之胺基酸序列； (2)  該第一重鏈包含與SEQ ID NO: 7所述之胺基酸序列具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、100%同一性之胺基酸序列；該第二重鏈包含與SEQ ID NO: 8所述之胺基酸序列具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、100%同一性之胺基酸序列；該輕鏈包含與SEQ ID NO: 9所述之胺基酸序列具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、100%同一性之胺基酸序列； (3)  該第一重鏈包含與SEQ ID NO: 3所述之胺基酸序列具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、100%同一性之胺基酸序列；該第二重鏈包含與SEQ ID NO: 10所述之胺基酸序列具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、100%同一性之胺基酸序列；該輕鏈包含與SEQ ID NO: 11所述之胺基酸序列具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、100%同一性之胺基酸序列； (4)  該第一重鏈包含與SEQ ID NO: 3所述之胺基酸序列具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、100%同一性之胺基酸序列；該第二重鏈包含與SEQ ID NO: 15所述之胺基酸序列具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、100%同一性之胺基酸序列；該輕鏈包含與SEQ ID NO: 16所述之胺基酸序列具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、100%同一性之胺基酸序列。 Example 56. A fusion protein of any one of Examples 46 to 55, comprising a first heavy chain, a second heavy chain, and two light chains respectively paired with the first heavy chain and the second heavy chain, wherein (1) the first heavy chain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence described in SEQ ID NO: 3; the second heavy chain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence described in SEQ ID NO: 5; the light chain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence described in SEQ ID NO: 5; (6) The amino acid sequence described in SEQ ID NO: 7 has an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% identity with the amino acid sequence described in SEQ ID NO: 7; (2) The first heavy chain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% identity with the amino acid sequence described in SEQ ID NO: 8; the second heavy chain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% identity with the amino acid sequence described in SEQ ID NO: 8; the light chain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% identity with the amino acid sequence described in SEQ ID NO: 8; 9. The amino acid sequence described herein has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% identity with the amino acid sequence described in SEQ ID NO: 3; (3) The first heavy chain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% identity with the amino acid sequence described in SEQ ID NO: 3; the second heavy chain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% identity with the amino acid sequence described in SEQ ID NO: 10; the light chain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% identity with the amino acid sequence described in SEQ ID NO: 10; The amino acid sequence described in SEQ ID NO: 3 has an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% identity with the amino acid sequence described in SEQ ID NO: 3; (4) The first heavy chain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% identity with the amino acid sequence described in SEQ ID NO: 3; the second heavy chain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% identity with the amino acid sequence described in SEQ ID NO: 15; the light chain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% identity with the amino acid sequence described in SEQ ID NO: 15; The amino acid sequence described in 16 has an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity.

實施例57.如實施例46至56中任一項之融合蛋白，其包含下列： (1)  具有SEQ ID NO: 3所述之胺基酸序列的該第一重鏈、具有SEQ ID NO: 5所述之胺基酸序列的該第二重鏈、及具有SEQ ID NO: 6所述之胺基酸序列的該輕鏈； (2)  具有SEQ ID NO: 7所述之胺基酸序列的該第一重鏈、具有SEQ ID NO: 8所述之胺基酸序列的該第二重鏈、及具有SEQ ID NO: 9所述之胺基酸序列的該輕鏈； (3)  具有SEQ ID NO: 3所述之胺基酸序列的該第一重鏈、具有SEQ ID NO: 10所述之胺基酸序列的該第二重鏈、及具有SEQ ID NO: 11所述之胺基酸序列的該輕鏈； (4)  具有SEQ ID NO: 3所述之胺基酸序列的該第一重鏈、具有SEQ ID NO: 15所述之胺基酸序列的該第二重鏈、及具有SEQ ID NO: 16所述之胺基酸序列的該輕鏈。 Example 57. A fusion protein of any one of Examples 46 to 56, comprising the following: (1) the first heavy chain having the amino acid sequence of SEQ ID NO: 3, the second heavy chain having the amino acid sequence of SEQ ID NO: 5, and the light chain having the amino acid sequence of SEQ ID NO: 6; (2) the first heavy chain having the amino acid sequence of SEQ ID NO: 7, the second heavy chain having the amino acid sequence of SEQ ID NO: 8, and the light chain having the amino acid sequence of SEQ ID NO: 9; (3) the first heavy chain having the amino acid sequence of SEQ ID NO: 3, the second heavy chain having the amino acid sequence of SEQ ID NO: 10, and the light chain having the amino acid sequence of SEQ ID NO: 11; (4) having SEQ ID NO: 3; The first heavy chain having the amino acid sequence described in SEQ ID NO: 3, the second heavy chain having the amino acid sequence described in SEQ ID NO: 15, and the light chain having the amino acid sequence described in SEQ ID NO: 16.

實施例58.如實施例46至57中任一項之融合蛋白，其中該融合蛋白以不大於20 nM、15 nM、10 nM、或5 nM之解離常數(KD)結合至FAP。Example 58. A fusion protein as described in any of Examples 46 to 57, wherein the fusion protein binds to FAP with a dissociation constant (KD) not greater than 20 nM, 15 nM, 10 nM, or 5 nM.

實施例59.如實施例46至58中任一項之融合蛋白，其中該融合蛋白以不大於20 nM、15 nM、10 nM、或5 nM之解離常數(KD)結合至LTßR。Example 59. A fusion protein as described in any of Examples 46 to 58, wherein the fusion protein binds to LTßR with a dissociation constant (KD) not greater than 20 nM, 15 nM, 10 nM, or 5 nM.

實施例60.如實施例46至59中任一項之融合蛋白，其中該融合蛋白幾乎不結合至人類或食蟹獼猴HVEM。Example 60. A fusion protein as described in any of Examples 46 to 59, wherein the fusion protein hardly binds to human or cynomolgus monkey HVEM.

實施例61.如實施例46至60中任一項之融合蛋白，其中融合蛋白中之該LIGHT突變體能夠降低與DcR3之結合親和力。Example 61. A fusion protein as described in any of Examples 46 to 60, wherein the LIGHT mutant of the fusion protein is capable of reducing the binding affinity to DcR3.

實施例62.如實施例46至61中任一項之融合蛋白，其中該融合蛋白特異性結合至人類FAP，且/或不結合至DPPIV。Example 62. A fusion protein as described in any of Examples 46 to 61, wherein the fusion protein specifically binds to human FAP and/or does not bind to DPPIV.

實施例63.一種經單離多核苷酸，其編碼如實施例39至45中任一項之抗FAP抗體或其抗原結合片段或如實施例46至62中任一項之融合蛋白的任何序列。Example 63. A single isolated polynucleotide encoding any sequence of an anti-FAP antibody or its antigen-binding fragment as described in any of Examples 39 to 45, or a fusion protein as described in any of Examples 46 to 62.

實施例64.一種構築體，其包含如實施例63之多核苷酸。Example 64. A structure comprising a polynucleotide as in Example 63.

實施例65.一種抗體表現系統，其包含包括如實施例63之經單離多核苷酸或具有整合有如實施例63之外源多核苷酸的基因體的該構築體，其中較佳地，該表現系統係細胞表現系統。Example 65. An antibody expression system comprising the construct including a single polynucleotide as in Example 63 or having a genotype integrating an exogenous polynucleotide as in Example 63, wherein preferably, the expression system is a cell expression system.

實施例66.一種用於產生如實施例39至45中任一項之抗FAP抗體或其抗原結合片段或如實施例46至62中任一項之融合蛋白的方法，其包含：在適合表現該抗體的條件下，使用如實施例65之抗體表現系統表現該抗體或融合蛋白。Example 66. A method for generating an anti-FAP antibody or an antigen-binding fragment thereof as described in any one of Examples 39 to 45, or a fusion protein as described in any one of Examples 46 to 62, comprising: expressing the antibody or fusion protein using an antibody expression system as described in Example 65 under conditions suitable for expressing the antibody.

實施例67.一種醫藥組成物，其包含如實施例39至45中任一項之抗FAP抗體或其抗原結合片段或如實施例46至62中任一項之融合蛋白、及醫藥上可接受之載劑。Example 67. A pharmaceutical composition comprising an anti-FAP antibody or an antigen-binding fragment thereof as in any of Examples 39 to 45 or a fusion protein as in any of Examples 46 to 62, and a pharmaceutically acceptable delivery vehicle.

實施例68.一種套組，其包含如實施例39至45中任一項之抗FAP抗體或其抗原結合片段、如實施例46至62中任一項之融合蛋白、如實施例63之經單離多核苷酸、或如實施例64之構築體。Example 68. A kit comprising an anti-FAP antibody or an antigen-binding fragment thereof as in any of Examples 39 to 45, a fusion protein as in any of Examples 46 to 62, a monoisopropyl nucleotide as in Example 63, or a building block as in Example 64.

實施例69.一種如實施例39至45中任一項之抗FAP抗體或其抗原結合片段、如實施例46至62中任一項之融合蛋白、或如實施例67之醫藥組成物於製造用於預防、診斷、或治療疾病、病症、或病況之治療劑的用途。Example 69. Use of an anti-FAP antibody or antigen-binding fragment thereof as described in any of Examples 39 to 45, a fusion protein as described in any of Examples 46 to 62, or a pharmaceutical composition as described in Example 67 in the manufacture of a treatment for the prevention, diagnosis, or treatment of a disease, symptom, or condition.

實施例70.如實施例69之用途，其中該疾病、病症、或病況包含腫瘤疾病。Example 70. The use of Example 69, wherein the disease, symptom, or condition includes a tumor.

實施例71.如實施例69或70之用途，其中至少一個腫瘤細胞表現FAP。Example 71. As used in Examples 69 or 70, wherein at least one tumor cell exhibits FAP.

實施例72.如實施例69至72中任一項之用途，其中該等腫瘤疾病係實體腫瘤。Example 72. As used in any of Examples 69 to 72, wherein the tumor disease is a solid tumor.

實施例73.如實施例69至72中任一項之用途，其中該腫瘤包含胃癌、肝癌、肺癌、結腸直腸癌、乳癌、前列腺癌、皮膚癌、骨癌、多發性骨髓瘤、神經膠質瘤、卵巢癌、胰臟癌、子宮頸癌、甲狀腺癌、喉癌、急性骨髓性白血病、慢性骨髓性白血病、急性淋巴母細胞白血病、慢性淋巴母細胞白血病、腦腫瘤、神經母細胞瘤、視網膜母細胞瘤、頭頸癌、唾液腺癌、及淋巴瘤。Example 73. As used in any of Examples 69 to 72, wherein the tumor includes gastric cancer, liver cancer, lung cancer, colorectal cancer, breast cancer, prostate cancer, skin cancer, bone cancer, multiple myeloma, glioma, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and lymphoma.

實施例74.一種用於治療有需要之對象的方法，其包含向該對象投予治療有效量的如實施例39至45中任一項之抗FAP抗體或其抗原結合片段、如實施例46至62中任一項之融合蛋白、或如實施例67之醫藥組成物。Example 74. A method for treating a subject in need, comprising administering to the subject a therapeutically effective amount of an anti-FAP antibody or its antigen-binding fragment, such as any of Examples 39 to 45, a fusion protein, such as any of Examples 46 to 62, or a pharmaceutical composition, such as Example 67.

實施例75.一種用於降低腫瘤生長速率或腫瘤細胞數目的方法，其包含使腫瘤細胞與有效量的如實施例39至45中任一項之抗FAP抗體或其抗原結合片段、如實施例46至62中任一項之融合蛋白、或如實施例67之醫藥組成物接觸。Example 75. A method for reducing tumor growth rate or tumor cell number, comprising contacting tumor cells with an effective amount of an anti-FAP antibody or its antigen-binding fragment as in any of Examples 39 to 45, a fusion protein as in any of Examples 46 to 62, or a pharmaceutical composition as in Example 67.

實施例76.一種殺滅腫瘤細胞之方法，其包含使腫瘤細胞與有效量的如實施例39至45中任一項之抗FAP抗體或其抗原結合片段、如實施例46至62中任一項之融合蛋白、或如實施例67之醫藥組成物接觸。 序列 Example 76. A method for killing tumor cells, comprising contacting the tumor cells with an effective amount of an anti-FAP antibody or its antigen-binding fragment as described in any of Examples 39 to 45, a fusion protein as described in any of Examples 46 to 62, or a pharmaceutical composition as described in Example 67. Sequence

下 表 5中的序列係如本文所述之FAP及其表位的例示性序列。 表5 ：FAP 及其表位的例示性胺基酸序列 SEQ ID NO: 描述 胺基酸序列 43 人類FAP (Uniprot Q12884) MKTWVKIVFGVATSAVLALLVMCIVLRPSRVHNSEENTMRALTLKDILNGTFSYKTFFPNWISGQEYLHQSADNNIVLYNIETGQSYTILSNRTMKSVNASNYGLSPDRQFVYLESDYSKLWRYSYTATYYIYDLSNGEFVRGNELPRPIQYLCWSPVGSKLAYVYQNNIYLKQRPGDPPFQITFNGRENKIFNGIPDWVYEEEMLATKYALWWSPNGKFLAYAEFNDTDIPVIAYSYYGDEQYPRTINIPYPKAGAKNPVVRIFIIDTTYPAYVGPQEVPVPAMIASSDYYFSWLTWVTDERVCLQWLKRVQNVSVLSICDFREDWQTWDCPKTQEHIEESRTGWAGGFFVSTPVFSYDAISYYKIFSDKDGYKHIHYIKDTVENAIQITSGKWEAINIFRVTQDSLFYSSNEFEEYPGRRNIYRISIGSYPPSKKCVTCHLRKERCQYYTASFSDYAKYYALVCYGPGIPISTLHDGRTDQEIKILEENKELENALKNIQLPKEEIKKLEVDEITLWYKMILPPQFDRSKKYPLLIQVYGGPCSQSVRSVFAVNWISYLASKEGMVIALVDGRGTAFQGDKLLYAVYRKLGVYEVEDQITAVRKFIEMGFIDEKRIAIWGWSYGGYVSSLALASGTGLFKCGIAVAPVSSWEYYASVYTERFMGLPTKDDNLEHYKNSTVMARAEYFRNVDYLLIHGTADDNVHFQNSAQIAKALVNAQVDFQAMWYSDQNHGLSGLSTNHLYTHMTHFLKQCFSLSD 44 食蟹獼猴FAP (NCBI XP_005573377.1) MKTWVKIVFGVATSAVLALLVMCIVLRPPRVHNSEENTMRALTLKDILNGTFSYKTFFPNWISGQEYLHQSADNNIVLYNIETGQSYTILSNRTMKSVNASNYGLSPDRQFVYLESDYSKLWRYSYTATYYIYDLSNGEFVRGNELPRPIQYLCWSPVGSKLAYVYQNNIYLKQRPGDPPFQITFNGRENKIFNGIPDWVYEEEMLATKYALWWSPNGKFLAYAEFNDTDIPVIAYSYYGDEQYPRTINIPYPKAGAKNPFVRIFIIDTTYPAYVGPQEVPVPAMIASSDYYFSWLTWVTDERVCLQWLKRVQNVSVLSICDFREDWQTWDCPKTQEHIEESRTGWAGGFFVSTPVFSYDAISYYKIFSDKDGYKHIHYIKDTVENAIQITSGKWEAINIFRVTQDSLFYSSNEFEDYPGRRNIYRISIGSYPPSKKCVTCHLRKERCQYYTASFSDYAKYYALVCYGPGIPISTLHDGRTDQEIKILEENKELENALKNIQLPKEEIKKLEVDEITLWYKMILPPQFDRSKKYPLLIQVYGGPCSQSVRSVFAVNWISYLASKEGMVIALVDGRGTAFQGDKLLYAVYRKLGVYEVEDQITAVRKFIEMGFIDEKRIAIWGWSYGGYVSSLALASGTGLFKCGIAVAPVSSWEYYASVYTERFMGLPTKDDNLEHYKNSTVMARAEYFRNVDYLLIHGTADDNVHFQNSAQIAKALVNAQVDFQAMWYSDQNHGLSGLSTNHLYTHMTHFLKQCFSLSD 45 小鼠FAP (Uniprot P97321) MKTWLKTVFGVTTLAALALVVICIVLRPSRVYKPEGNTKRALTLKDILNGTFSYKTYFPNWISEQEYLHQSEDDNIVFYNIETRESYIILSNSTMKSVNATDYGLSPDRQFVYLESDYSKLWRYSYTATYYIYDLQNGEFVRGYELPRPIQYLCWSPVGSKLAYVYQNNIYLKQRPGDPPFQITYTGRENRIFNGIPDWVYEEEMLATKYALWWSPDGKFLAYVEFNDSDIPIIAYSYYGDGQYPRTINIPYPKAGAKNPVVRVFIVDTTYPHHVGPMEVPVPEMIASSDYYFSWLTWVSSERVCLQWLKRVQNVSVLSICDFREDWHAWECPKNQEHVEESRTGWAGGFFVSTPAFSQDATSYYKIFSDKDGYKHIHYIKDTVENAIQITSGKWEAIYIFRVTQDSLFYSSNEFEGYPGRRNIYRISIGNSPPSKKCVTCHLRKERCQYYTASFSYKAKYYALVCYGPGLPISTLHDGRTDQEIQVLEENKELENSLRNIQLPKVEIKKLKDGGLTFWYKMILPPQFDRSKKYPLLIQVYGGPCSQSVKSVFAVNWITYLASKEGIVIALVDGRGTAFQGDKFLHAVYRKLGVYEVEDQLTAVRKFIEMGFIDEERIAIWGWSYGGYVSSLALASGTGLFKCGIAVAPVSSWEYYASIYSERFMGLPTKDDNLEHYKNSTVMARAEYFRNVDYLLIHGTADDNVHFQNSAQIAKALVNAQVDFQAMWYSDQNHGISSGRSQNHLYTHMTHFLKQCFSLSD The sequences in Table 5 below are exemplary sequences of FAP and its epitopes as described herein. Table 5 : Exemplary amino acid sequences of FAP and its epitopes SEQ ID NO: describe amino acid sequence 43 Human FAP (Uniprot Q12884) MKTWVKIVFGVATSAVLALLVMCIVLRPSRVHNSEENTMRALTLKDILNGTFSYKTFFPNWISGQEYLHQSADNNIVLYNIETGQSYTILSNRTMKSVNASNYGLSPDRQFVYLESDYSKLWRYSYTATYYIYDLSNGEFVRGNELPRPIQYLCWSPVGSKLAYVYQNNIYLKQRPGDPPFQITFNGREN KIFNGIPDWVYEEEMLATKYALWWSPNGKFLAYAEFNDTDIPVIAYSYYGDEQYPRTINIPYPKAGAKNPVVRIFIIDTTYPAYVGPQEVPVPAMIASSDYYFSWLTWVTDERVCLQWLKRVQNVSVLSICDFREDWQTWDCPKTQEHIEESRTGWAGGFFVSTPVFSYDAISYYKIFSDKDGYKHIHYI KDTVENAIQITSGKWEAINIFRVTQDSLFYSSNEFEEYPGRRNIYRISIGSYPPSKKCVTCHLRKERCQYYTASFSDYAKYYALVCYGPGIPISTLHDGRTDQEIKILEENKELENALKNIQLPKEEIKKLEVDEITLWYKMILPPQFDRSKKYPLLIQVYGGPCSQSVRSVFAVNWISYLASKEGMVIA LVDGRGGTAFQGDKLLYAVYRKLGVYEVEDQITAVRKFIEMGFIDEKRIAIWGWSYGGYVSSLALASGTGLFKCGIAVAPVSSWEYYASVYTERFMGLPTKDDNLEHYKNSTVMARAEYFRNVDYLLIHGTADDNVHFQNSAQIAKALVNAQVDFQAMWYSDQNHGLSGSTNHLYTHMTHFLKQCFSLSD 44 Crab-eating macaque (FAP) (NCBI XP_005573377.1) MKTWVKIVFGVATSAVLALLVMCIVLRPPRVHNSEENTMRALTLKDILNGTFSYKTFFPNWISGQEYLHQSADNNIVLYNIETGQSYTILSNRTMKSVNASNYGLSPDRQFVYLESDYSKLWRYSYTATYYIYDLSNGEFVRGNELPRPIQYLCWSPVGSKLAYVYQNNIYLKQRPGDPPFQITFNGREN KIFNGIPDWVYEEEMLATKYALWWSPNGKFLAYAEFNDTDIPVIAYSYYGDEQYPRTINIPYPKAGAKNPFVRIFIIDTTYPAYVGPQEVPVPAMIASSDYYFSWLTWVTDERVCLQWLKRVQNVSVLSICDFREDWQTWDCPKTQEHIEESRTGWAGGFFVSTPVFSYDAISYYKIFSDKDGYKHIHYI KDTVENAIQITSGKWEAINIFRVTQDSLFYSSNEFEDYPGRRNIYRISIGSYPPSKKCVTCHLRKERCQYYTASFSDYAKYYALVCYGPGIPISTLHDGRTDQEIKILEENKELENALKNIQLPKEEIKKLEVDEITLWYKMILPPQFDRSKKYPLLIQVYGGPCSQSVRSVFAVNWISYLASKEGMVIA LVDGRGGTAFQGDKLLYAVYRKLGVYEVEDQITAVRKFIEMGFIDEKRIAIWGWSYGGYVSSLALASGTGLFKCGIAVAPVSSWEYYASVYTERFMGLPTKDDNLEHYKNSTVMARAEYFRNVDYLLIHGTADDNVHFQNSAQIAKALVNAQVDFQAMWYSDQNHGLSGSTNHLYTHMTHFLKQCFSLSD 45 Mouse FAP (Uniprot P97321) MKTWLKTVFGVTTLAALALVVICIVLRPSRVYKPEGNTKRALTLKDILNGTFSYKTYFPNWISEQEYLHQSEDDNIVFYNIETRESYIILSNSTMKSVNATDYGLSPDRQFVYLESDYSKLWRYSYTATYYIYDLQNGEFVRGYELPRPIQYLCWSPVGSKLAYVYQNNIYLKQRPGDPPFQITYTGREN RIFNGIPDWVYEEEMLATKYALWWSPDGKFLAYVEFNDSDIPIIAYSYYGDGQYPRTINIPYPKAGAKNPVVRVFIVDTTYPHHVGPMEVPVPEMIASSDYYFSWLTWVSSERVCLQWLKRVQNVSVLSICDFREDWHAWECPKNQEHVEESRTGWAGGFFVSTPAFSQDATSYYKIFSDKDGYKHIHYI KDTVENAIQITSGKWEAIYIFRVTQDSLFYSSNEFEGYPGRRNIYRISIGNSPSPSKKCVTCHLRKERCQYYTASFSYKAKYYALVCYGPGLPISTLHDGRTDQEIQVLEENKELENSLRNIQLPKVEIKKLKDGGLTFWYKMILPPQFDRSKKYPLLIQVYGGPCSQSVKSVFAVNWITYLASKEGIVIA LVDGRGGTAFQGDKFLHAVYRKLGVYEVEDQLTAVRKFIEMGFIDEERIAIWGWSYGGYVSSLALASGTGFKCGIAVAPVSSWEYYASIYSERFMGLPTKDDNLEHYKNSTVMARAEYFRNVDYLLIHGTADDNVHFQNSAQIAKALVNAQVDFQAMWYSDQNHGISSGRSQNHLYTHMTHFLKQCFSLSD

下 表 5.1中的序列係如本文所述之腫瘤相關細胞受體(TACR)及其表位的例示性序列。 表5.1 ：TACR 及其表位的例示性胺基酸序列 SEQ ID NO: 描述 胺基酸序列 49 人類LTβR (Uniprot P36941) MLLPWATSAPGLAWGPLVLGLFGLLAASQPQAVPPYASENQTCRDQEKEYYEPQHRICCSRCPPGTYVSAKCSRIRDTVCATCAENSYNEHWNYLTICQLCRPCDPVMGLEEIAPCTSKRKTQCRCQPGMFCAAWALECTHCELLSDCPPGTEAELKDEVGKGNNHCVPCKAGHFQNTSSPSARCQPHTRCENQGLVEAAPGTAQSDTTCKNPLEPLPPEMSGTMLMLAVLLPLAFFLLLATVFSCIWKSHPSLCRKLGSLLKRRPQGEGPNPVAGSWEPPKAHPYFPDLVQPLLPISGDVSPVSTGLPAAPVLEAGVPQQQSPLDLTREPQLEPGEQSQVAHGTNGIHVTGGSMTITGNIYIYNGPVLGGPPGPGDLPATPEPPYPIPEEGDPGPPGLSTPHQEDGKAWHLAETEHCGATPSNRGPRNQFITHD 50 食蟹獼猴LTβR (Uniprot A0A2K5VGQ6) MRLPWATSAPGLAWGPLVLGLFGLLAASQPQVVRKGPVPPYGSENQTCRDQEKEYYEPRHRICCSRCPPGTYVSAKCSRSRDTVCATCAENSYNEHWNYLTICQLCRPCDPVMGLEEIAPCTSKRKTQCRCQPGMFCAAWALECTHCELLSDCPPGTEAELKDEVGKGNNHCVPCKAGHFQNTSSPSARCQPHTRCEDQGLVEAAPGTAQSDTTCRNPSESLPPEMSGTMLMLAILLPLAFFLLLATIFACIWKSHPSLCRKLGSLLKRHPQGEGPNPVAAGRDPPKANPQYPDLVEPLLPISGDVSPVSTGLPTALVSEEGVPQQQSPLDLTTEPQLEPGEQNQVAHGTNGIHVTGGSMTITGNIYIYNGPVLGGPPGPGDLPATPDPPYPIPEEGDPGPPGLSTPHQEDGKAWHLAETEHCGATPSNRGPRSQFITYD 51 小鼠LTβR (Uniprot P50284) MRLPRASSPCGLAWGPLLLGLSGLLVASQPQLVPPYRIENQTCWDQDKEYYEPMHDVCCSRCPPGEFVFAVCSRSQDTVCKTCPHNSYNEHWNHLSTCQLCRPCDIVLGFEEVAPCTSDRKAECRCQPGMSCVYLDNECVHCEEERLVLCQPGTEAEVTDEIMDTDVNCVPCKPGHFQNTSSPRARCQPHTRCEIQGLVEAAPGTSYSDTICKNPPEPGAMLLLAILLSLVLFLLFTTVLACAWMRHPSLCRKLGTLLKRHPEGEESPPCPAPRADPHFPDLAEPLLPMSGDLSPSPAGPPTAPSLEEVVLQQQSPLVQARELEAEPGEHGQVAHGANGIHVTGGSVTVTGNIYIYNGPVLGGTRGPGDPPAPPEPPYPTPEEGAPGPSELSTPYQEDGKAWHLAETETLGCQDL The sequences in Table 5.1 below are illustrative sequences of tumor-associated cell receptors (TACRs) and their epitopes as described herein. Table 5.1 : Illustrative amino acid sequences of TACRs and their epitopes SEQ ID NO: describe amino acid sequence 49 Human LTβR (Uniprot P36941) MLLPWATSAPGLAWGPLVLGLFGLLAASQPQAVPPYASENQTCRDQEKEYYEPQHRICCSRCPPGTYVSAKCSRIRDTVCATCAENSYNEHWNYLTICQLCRPCDPVM GLEEIAPCTSKRKTQCRCQPGMFCAAWALECTHCELLSDCPPGTEAELKDEVGKGNNHCVPCKAGHFQNTSSPSARCQPHTRCENQGLVEAAPGTAQSDTTCKNPLEPL PPEMSGTMLMLAVLLPLAFFLLLATVFSCIWKSHPSLCRKLGSLLKRRPQGEGPNPVAGSWEPPKAHPYFPDLVQPLLPISGDVSPVSTGLPAAPVLEAGVPQQQSPLD LTREPQLEPGEQSQVAHGTNGIHVTGGSMTITGNIYIYNGPVLGGPPGPGDLPATPEPPYPIPEEGDPGPPGLSTPHQEDGKAWHLAETEHCGATPSNRGPRNQFITHD 50 Crab-eating macaque LTβR (Uniprot A0A2K5VGQ6) MRLPWATSAPGLAWGPLVLGLFGLLAASQPQVVRKGPVPPYGSENQTCRDQEKEYYEPRHRICCSRCPPGTYVSAKCSRSRDTVCATCAENSYNEHWNYLTICQLCRPCD PVMGLEEIAPCTSKRKTQCRCQPGMFCAAWALECTHCELLSDCPPGTEAELKDEVGKGNNHCVPCKAGHFQNTSSPSARCQPHTRCEDQGLVEAAPGTAQSDTTCRNPSE SLPPEMSGTMLMLAILLPLAFFLLLATIFACIWKSHPSLCRKLGSLLKRHPQGEGPNPVAAGRDPPKANPQYPDLVEPLLPISGDVSPVSTGLPTALVSEEGVPQQQSPL DLTTEPQLEPGEQNQVAHGTNGIHVTGGSMTITGNIYIYNGPVLGGPPGPGDLPATPDPPYPIPEEGDPGPPGLSTPHQEDGKAWHLAETEHCGATPSNRGPRSQFITYD 51 Mouse LTβR (Uniprot P50284) MRLPRASSPCGLAWGPLLLGLSGLLVASQPQLVPPYRIENQTCWDQDKEYYEPMHDVCCSRCPPGEFVFAVCSRSQDTVCKTCPHNSYNEHWNHLSTCQLCRP CDIVLGFEEVAPCTSDRKAAECRCQPGMSCVYLDNECVHCEEERLVLCQPGTEAEVTDEIMDTDVNCVPCKPGHFQNTSSPRARCQPHTRCEIQGLVEAAPGTSY SDTICKNPPEPGAMLLLAILLSLVLFLLFTTVLACAWMRHPSLCRKLGTLLKRHPEGEESPPCPAPRADPHFPDLAEPLLPMSGDLSPSPAGPPTAPSLEEVVLQQQSPLVQARELEAEPGEHGQVAHGANGIHVTGGSVTVTGNIYIYNGPVLGGTRGPGDPPAPPEPPYPTPEEGAPGPSELSTPYQEDGKAWHLAETETLGCQDL

下 表 6中的序列繪示可用以產生多肽序列（例如抗FAP抗體、雙特異性抗體、或其融合蛋白）及/或系統及組成物且執行本文所述方法的共有胺基酸序列。在 表 6中，括號表示替代的胺基酸，例如[Y/T]意指Y或T。在另一實例中，[Y/N/K]意指Y、N、或K。此外，在 表 6中，下標數字表示括號中所指示的胺基酸的實例，例如[R] 0-1意指沒有R或單一R。根據需要，可容易地從胺基酸序列推導出核苷酸序列。 表6 ：例示性共有胺基酸序列 SEQ ID NO: 描述 序列 AB 共有 26 HCDR1 IYGVN 9E3嵌合體(ABC1139)、ABC1931、ABC1930、ABC1947、ABC2066、ABC2067、及ABC2097 27 HCDR2 AIWSGGRKDYX 2LSLKS 9E3嵌合體(ABC1139)、ABC1931、ABC1930、ABC1947、ABC2066、ABC2067、及ABC2097 28 HCDR3 SQDMPGYFDY 9E3嵌合體(ABC1139)、ABC1931、ABC1930、ABC1947、ABC2066、ABC2067、及ABC2097 23 LCDR1 KTNQNVDYX 1GNTFMH 9E3嵌合體(ABC1139)、ABC1931、ABC1930、ABC1947、ABC2066、ABC2067、及ABC2097 24 LCDR2 LASNLAS 9E3嵌合體(ABC1139)、ABC1931、ABC1930、ABC1947、ABC2066、ABC2067、及ABC2097 25 LCDR3 QQSRNLPYT 9E3嵌合體(ABC1139)、ABC1931、ABC1930、ABC1947、ABC2066、ABC2067、及ABC2097 X 1係N或S；X 2係N或S The sequences in Table 6 below illustrate common amino acid sequences that can be used to generate polypeptide sequences (e.g., anti-FAP antibodies, bispecific antibodies, or fusion proteins thereof) and/or systems and compositions and to perform the methods described herein. In Table 6 , parentheses indicate substituted amino acids, for example, [Y/T] means Y or T. In another example, [Y/N/K] means Y, N, or K. Furthermore, in Table 6 , subscript numbers indicate instances of the amino acid indicated in parentheses, for example, [R] 0-1 means no R or a single R. Nucleotide sequences can be readily derived from amino acid sequences as needed. Table 6 : Illustrative Common Amino Acid Sequences SEQ ID NO: describe sequence A and B have a total of 26 HCDR1 IYGVN 9E3 chimera (ABC1139), ABC1931, ABC1930, ABC1947, ABC2066, ABC2067, and ABC2097 27 HCDR2 AIWSGGRKDYX 2 LSLKS 9E3 chimera (ABC1139), ABC1931, ABC1930, ABC1947, ABC2066, ABC2067, and ABC2097 28 HCDR3 SQDMPGYFDY 9E3 chimera (ABC1139), ABC1931, ABC1930, ABC1947, ABC2066, ABC2067, and ABC2097 twenty three LCDR1 KTNQNVDYX 1 GNTFMH 9E3 chimera (ABC1139), ABC1931, ABC1930, ABC1947, ABC2066, ABC2067, and ABC2097 twenty four LCDR2 LASNLAS 9E3 chimera (ABC1139), ABC1931, ABC1930, ABC1947, ABC2066, ABC2067, and ABC2097 25 LCDR3 QQSRNLPYT 9E3 chimera (ABC1139), ABC1931, ABC1930, ABC1947, ABC2066, ABC2067, and ABC2097 X1 is either N or S; X2 is either N or S

下 表 7中的序列繪示可用以產生本揭露的FAP結合劑（例如抗FAP抗體、雙特異性抗體、或其融合蛋白）及/或系統及組成物且執行本文所述方法的例示性CDR胺基酸序列。根據需要，可容易地從胺基酸序列推導出核苷酸序列。 表7 ：例示性CDR 胺基酸序列 AB 描述 HC LC CDRH1 CDRH2 CDRH3 CDRL1 CDRL2 CDRL3 9E3 嵌合體(ABC1139) Chothia GFSLSIY (SEQ ID NO: 52) SGG (SEQ ID NO: 58) QDMPGYFD (SEQ ID NO: 67) NQNVDYNGNTF (SEQ ID NO: 73) LAS (SEQ ID NO: 83) SRNLPY (SEQ ID NO: 88) Honegger VSGFSLSIYG (SEQ ID NO: 53) IWSGGRKDYNLSLKSR (SEQ ID NO: 59) SQDMPGYFD (SEQ ID NO: 68) TNQNVDYNGNTF (SEQ ID NO: 74) LASNLASGIPDR (SEQ ID NO: 84) SRNLPY (SEQ ID NO: 88) IMGT GFSLSIYG (SEQ ID NO: 54) IWSGGRK (SEQ ID NO: 60) ARSQDMPGYFDY (SEQ ID NO: 69) QNVDYNGNTF (SEQ ID NO: 75) LAS (SEQ ID NO: 83) QQSRNLPYT (SEQ ID NO: 25) Kabat IYGVN (SEQ ID NO: 26) AIWSGGRKDYNLSLKS (SEQ ID NO: 61) SQDMPGYFDY (SEQ ID NO: 28) KTNQNVDYNGNTFMH (SEQ ID NO: 76) LASNLAS (SEQ ID NO: 24) QQSRNLPYT (SEQ ID NO: 25) ABC1931 Chothia GFSLSIY (SEQ ID NO: 52) SGG (SEQ ID NO: 58) QDMPGYFD (SEQ ID NO: 67) TNQNVDYSGNTF (SEQ ID NO: 77) LAS (SEQ ID NO: 83) SRNLPY (SEQ ID NO: 88) Honegger VSGFSLSIYG (SEQ ID NO: 53) IWSGGRKDYSLSLKSR (SEQ ID NO: 62) SQDMPGYFD (SEQ ID NO: 68) TNQNVDYSGNTF (SEQ ID NO: 77) LASNLASGIPER (SEQ ID NO: 85) SRNLPY (SEQ ID NO: 88) IMGT GFSLSIYG (SEQ ID NO: 54) IWSGGRK (SEQ ID NO: 60) ARSQDMPGYFDY (SEQ ID NO: 69) NQNVDYSGNTF (SEQ ID NO: 78) LAS (SEQ ID NO: 83) QQSRNLPYT (SEQ ID NO: 25) Kabat IYGVN (SEQ ID NO: 26) AIWSGGRKDYSLSLKS (SEQ ID NO: 63) SQDMPGYFDY (SEQ ID NO: 28) KTNQNVDYSGNTFMH (SEQ ID NO: 79) LASNLAS (SEQ ID NO: 24) QQSRNLPYT (SEQ ID NO: 25) ABC1930 Chothia GFSLSIY (SEQ ID NO: 52) SGG (SEQ ID NO: 58) QDMPGYFD (SEQ ID NO: 67) TNQNVDYSGNTF (SEQ ID NO: 77) LAS (SEQ ID NO: 83) SRNLPY (SEQ ID NO: 88) Honegger VSGFSLSIYG (SEQ ID NO: 53) IWSGGRKDYSLSLKSR (SEQ ID NO: 62) SQDMPGYFD (SEQ ID NO: 68) TNQNVDYSGNTF (SEQ ID NO: 77) LASNLASGIPER (SEQ ID NO: 85) SRNLPY (SEQ ID NO: 88) IMGT GFSLSIYG (SEQ ID NO: 54) IWSGGRK (SEQ ID NO: 60) ARSQDMPGYFDY (SEQ ID NO: 69) NQNVDYSGNTF (SEQ ID NO: 78) LAS (SEQ ID NO: 83) QQSRNLPYT (SEQ ID NO: 25) Kabat IYGVN (SEQ ID NO: 26) AIWSGGRKDYSLSLKS (SEQ ID NO: 63) SQDMPGYFDY (SEQ ID NO: 28) KTNQNVDYSGNTFMH (SEQ ID NO: 79) LASNLAS (SEQ ID NO: 24) QQSRNLPYT (SEQ ID NO: 25) ABC1773 Chothia GFSLSTAGM (SEQ ID NO: 55) WDD (SEQ ID NO: 64) MIFNFYFD (SEQ ID NO: 70) SSRVGY (SEQ ID NO: 80) DTS (SEQ ID NO: 86) GSGYPF (SEQ ID NO: 89) Honegger FSGFSLSTAGMS (SEQ ID NO: 56) IWWDDKKHYNPSLKDR (SEQ ID NO: 65) DMIFNFYFD (SEQ ID NO: 71) ASSRVGY (SEQ ID NO: 81) DTSKLASGVPSR (SEQ ID NO: 87) GSGYPF (SEQ ID NO: 89) IMGT GFSLSTAGMS (SEQ ID NO: 57) IWWDDKK (SEQ ID NO: 66) ARDMIFNFYFDV (SEQ ID NO: 72) SRVGY (SEQ ID NO: 82) DTS (SEQ ID NO: 86) FQGSGYPFT (SEQ ID NO: 31) Kabat TAGMSVG (SEQ ID NO: 32) DIWWDDKKHYNPSLKD (SEQ ID NO: 33) DMIFNFYFDV (SEQ ID NO: 34) SASSRVGYMH (SEQ ID NO: 29) DTSKLAS (SEQ ID NO: 30) FQGSGYPFT (SEQ ID NO: 31) ABC1947 Chothia GFSLSIY (SEQ ID NO: 52) SGG (SEQ ID NO: 58) QDMPGYFD (SEQ ID NO: 67) TNQNVDYSGNTF (SEQ ID NO: 77) LAS (SEQ ID NO: 83) SRNLPY (SEQ ID NO: 88) Honegger VSGFSLSIYG (SEQ ID NO: 53) IWSGGRKDYSLSLKSR (SEQ ID NO: 62) SQDMPGYFD (SEQ ID NO: 68) TNQNVDYSGNTF (SEQ ID NO: 77) LASNLASGIPER (SEQ ID NO: 85) SRNLPY (SEQ ID NO: 88) IMGT GFSLSIYG (SEQ ID NO: 54) IWSGGRK (SEQ ID NO: 60) ARSQDMPGYFDY (SEQ ID NO: 69) NQNVDYSGNTF (SEQ ID NO: 78) LAS (SEQ ID NO: 83) QQSRNLPYT (SEQ ID NO: 25) Kabat IYGVN (SEQ ID NO: 26) AIWSGGRKDYSLSLKS (SEQ ID NO: 63) SQDMPGYFDY (SEQ ID NO: 28) KTNQNVDYSGNTFMH (SEQ ID NO: 79) LASNLAS (SEQ ID NO: 24) QQSRNLPYT (SEQ ID NO: 25) ABC2066 Chothia GFSLSIY (SEQ ID NO: 52) SGG (SEQ ID NO: 58) QDMPGYFD (SEQ ID NO: 67) TNQNVDYSGNTF (SEQ ID NO: 77) LAS (SEQ ID NO: 83) SRNLPY (SEQ ID NO: 88) Honegger VSGFSLSIYG (SEQ ID NO: 53) IWSGGRKDYSLSLKSR (SEQ ID NO: 62) SQDMPGYFD (SEQ ID NO: 68) TNQNVDYSGNTF (SEQ ID NO: 77) LASNLASGIPER (SEQ ID NO: 85) SRNLPY (SEQ ID NO: 88) IMGT GFSLSIYG (SEQ ID NO: 54) IWSGGRK (SEQ ID NO: 60) ARSQDMPGYFDY (SEQ ID NO: 69) NQNVDYSGNTF (SEQ ID NO: 78) LAS (SEQ ID NO: 83) QQSRNLPYT (SEQ ID NO: 25) Kabat IYGVN (SEQ ID NO: 26) AIWSGGRKDYSLSLKS (SEQ ID NO: 63) SQDMPGYFDY (SEQ ID NO: 28) KTNQNVDYSGNTFMH (SEQ ID NO: 79) LASNLAS (SEQ ID NO: 24) QQSRNLPYT (SEQ ID NO: 25) ABC2067 Chothia GFSLSIY (SEQ ID NO: 52) SGG (SEQ ID NO: 58) QDMPGYFD (SEQ ID NO: 67) TNQNVDYSGNTF (SEQ ID NO: 77) LAS (SEQ ID NO: 83) SRNLPY (SEQ ID NO: 88) Honegger VSGFSLSIYG (SEQ ID NO: 53) IWSGGRKDYSLSLKSR (SEQ ID NO: 62) SQDMPGYFD (SEQ ID NO: 68) TNQNVDYSGNTF (SEQ ID NO: 77) LASNLASGIPER (SEQ ID NO: 85) SRNLPY (SEQ ID NO: 88) IMGT GFSLSIYG (SEQ ID NO: 54) IWSGGRK (SEQ ID NO: 60) ARSQDMPGYFDY (SEQ ID NO: 69) NQNVDYSGNTF (SEQ ID NO: 78) LAS (SEQ ID NO: 83) QQSRNLPYT (SEQ ID NO: 25) Kabat IYGVN (SEQ ID NO: 26) AIWSGGRKDYSLSLKS (SEQ ID NO: 63) SQDMPGYFDY (SEQ ID NO: 28) KTNQNVDYSGNTFMH (SEQ ID NO: 79) LASNLAS (SEQ ID NO: 24) QQSRNLPYT (SEQ ID NO: 25) ABC2097 Chothia GFSLSIY (SEQ ID NO: 52) SGG (SEQ ID NO: 58) QDMPGYFD (SEQ ID NO: 67) TNQNVDYSGNTF (SEQ ID NO: 77) LAS (SEQ ID NO: 83) SRNLPY (SEQ ID NO: 88) Honegger VSGFSLSIYG (SEQ ID NO: 53) IWSGGRKDYSLSLKSR (SEQ ID NO: 62) SQDMPGYFD (SEQ ID NO: 68) TNQNVDYSGNTF (SEQ ID NO: 77) LASNLASGIPER (SEQ ID NO: 85) SRNLPY (SEQ ID NO: 88) IMGT GFSLSIYG (SEQ ID NO: 54) IWSGGRK (SEQ ID NO: 60) ARSQDMPGYFDY (SEQ ID NO: 69) NQNVDYSGNTF (SEQ ID NO: 78) LAS (SEQ ID NO: 83) QQSRNLPYT (SEQ ID NO: 25) Kabat IYGVN (SEQ ID NO: 26) AIWSGGRKDYSLSLKS (SEQ ID NO: 63) SQDMPGYFDY (SEQ ID NO: 28) KTNQNVDYSGNTFMH (SEQ ID NO: 79) LASNLAS (SEQ ID NO: 24) QQSRNLPYT (SEQ ID NO: 25) The sequences illustrated in Table 7 below are exemplary CDR amino acid sequences that can be used to generate the FAP binding agents (e.g., anti-FAP antibodies, bispecific antibodies, or fusion proteins thereof) and/or systems and compositions disclosed herein and to perform the methods described herein. Nucleotide sequences can be readily deduced from amino acid sequences if needed. Table 7 : Exemplary CDR Amino Acid Sequences AB describe HC LC CDRH1 CDRH2 CDRH3 CDRL1 CDRL2 CDRL3 9E3 chimera (ABC1139) Chothia GFSLSIY (SEQ ID NO: 52) SGG (SEQ ID NO: 58) QDMPGYFD (SEQ ID NO: 67) NQNVDYNGNTF (SEQ ID NO: 73) LAS (SEQ ID NO: 83) SRNLPY (SEQ ID NO: 88) Honegger VSGFSLSIYG (SEQ ID NO: 53) IWSGGRKDYNLSLKSR (SEQ ID NO: 59) SQDMPGYFD (SEQ ID NO: 68) TNQNVDYNGNTF (SEQ ID NO: 74) LASNLASGIPDR (SEQ ID NO: 84) SRNLPY (SEQ ID NO: 88) IMGT GFSLSIYG (SEQ ID NO: 54) IWSGGRK (SEQ ID NO: 60) ARSQDMPGYFDY (SEQ ID NO: 69) QNVDYNGNTF (SEQ ID NO: 75) LAS (SEQ ID NO: 83) QQSRNLPYT (SEQ ID NO: 25) Kabat IYGVN (SEQ ID NO: 26) AIWSGGRKDYNLSLKS (SEQ ID NO: 61) SQDMPGYFDY (SEQ ID NO: 28) KTNQNVDYNGNTFMH (SEQ ID NO: 76) LASNLAS (SEQ ID NO: 24) QQSRNLPYT (SEQ ID NO: 25) ABC1931 Chothia GFSLSIY (SEQ ID NO: 52) SGG (SEQ ID NO: 58) QDMPGYFD (SEQ ID NO: 67) TNQNVDYSGNTF (SEQ ID NO: 77) LAS (SEQ ID NO: 83) SRNLPY (SEQ ID NO: 88) Honegger VSGFSLSIYG (SEQ ID NO: 53) IWSGGRKDYSLSLKSR (SEQ ID NO: 62) SQDMPGYFD (SEQ ID NO: 68) TNQNVDYSGNTF (SEQ ID NO: 77) LASNLASGIPER (SEQ ID NO: 85) SRNLPY (SEQ ID NO: 88) IMGT GFSLSIYG (SEQ ID NO: 54) IWSGGRK (SEQ ID NO: 60) ARSQDMPGYFDY (SEQ ID NO: 69) NQNVDYSGNTF (SEQ ID NO: 78) LAS (SEQ ID NO: 83) QQSRNLPYT (SEQ ID NO: 25) Kabat IYGVN (SEQ ID NO: 26) AIWSGGRKDYSLSLKS (SEQ ID NO: 63) SQDMPGYFDY (SEQ ID NO: 28) KTNQNVDYSGNTFMH (SEQ ID NO: 79) LASNLAS (SEQ ID NO: 24) QQSRNLPYT (SEQ ID NO: 25) ABC1930 Chothia GFSLSIY (SEQ ID NO: 52) SGG (SEQ ID NO: 58) QDMPGYFD (SEQ ID NO: 67) TNQNVDYSGNTF (SEQ ID NO: 77) LAS (SEQ ID NO: 83) SRNLPY (SEQ ID NO: 88) Honegger VSGFSLSIYG (SEQ ID NO: 53) IWSGGRKDYSLSLKSR (SEQ ID NO: 62) SQDMPGYFD (SEQ ID NO: 68) TNQNVDYSGNTF (SEQ ID NO: 77) LASNLASGIPER (SEQ ID NO: 85) SRNLPY (SEQ ID NO: 88) IMGT GFSLSIYG (SEQ ID NO: 54) IWSGGRK (SEQ ID NO: 60) ARSQDMPGYFDY (SEQ ID NO: 69) NQNVDYSGNTF (SEQ ID NO: 78) LAS (SEQ ID NO: 83) QQSRNLPYT (SEQ ID NO: 25) Kabat IYGVN (SEQ ID NO: 26) AIWSGGRKDYSLSLKS (SEQ ID NO: 63) SQDMPGYFDY (SEQ ID NO: 28) KTNQNVDYSGNTFMH (SEQ ID NO: 79) LASNLAS (SEQ ID NO: 24) QQSRNLPYT (SEQ ID NO: 25) ABC1773 Chothia GFSLSTAGM (SEQ ID NO: 55) WDD (SEQ ID NO: 64) MIFNFYFD (SEQ ID NO: 70) SSRVGY (SEQ ID NO: 80) DTS (SEQ ID NO: 86) GSGYPF (SEQ ID NO: 89) Honegger FSGFSLSTAGMS (SEQ ID NO: 56) IWWDDKKHYNPSLKDR (SEQ ID NO: 65) DMIFNFYFD (SEQ ID NO: 71) ASSRVGY (SEQ ID NO: 81) DTSKLASGVPSR (SEQ ID NO: 87) GSGYPF (SEQ ID NO: 89) IMGT GFSLSTAGMS (SEQ ID NO: 57) IWWDDKK (SEQ ID NO: 66) ARDMIFNFYFDV (SEQ ID NO: 72) SRVGY (SEQ ID NO: 82) DTS (SEQ ID NO: 86) FQGSGYPFT (SEQ ID NO: 31) Kabat TAGMSVG (SEQ ID NO: 32) DIWWDDKKHYNPSLKD (SEQ ID NO: 33) DMIFNFYFDV (SEQ ID NO: 34) SASSRVGYMH (SEQ ID NO: 29) DTSKLAS (SEQ ID NO: 30) FQGSGYPFT (SEQ ID NO: 31) ABC1947 Chothia GFSLSIY (SEQ ID NO: 52) SGG (SEQ ID NO: 58) QDMPGYFD (SEQ ID NO: 67) TNQNVDYSGNTF (SEQ ID NO: 77) LAS (SEQ ID NO: 83) SRNLPY (SEQ ID NO: 88) Honegger VSGFSLSIYG (SEQ ID NO: 53) IWSGGRKDYSLSLKSR (SEQ ID NO: 62) SQDMPGYFD (SEQ ID NO: 68) TNQNVDYSGNTF (SEQ ID NO: 77) LASNLASGIPER (SEQ ID NO: 85) SRNLPY (SEQ ID NO: 88) IMGT GFSLSIYG (SEQ ID NO: 54) IWSGGRK (SEQ ID NO: 60) ARSQDMPGYFDY (SEQ ID NO: 69) NQNVDYSGNTF (SEQ ID NO: 78) LAS (SEQ ID NO: 83) QQSRNLPYT (SEQ ID NO: 25) Kabat IYGVN (SEQ ID NO: 26) AIWSGGRKDYSLSLKS (SEQ ID NO: 63) SQDMPGYFDY (SEQ ID NO: 28) KTNQNVDYSGNTFMH (SEQ ID NO: 79) LASNLAS (SEQ ID NO: 24) QQSRNLPYT (SEQ ID NO: 25) ABC2066 Chothia GFSLSIY (SEQ ID NO: 52) SGG (SEQ ID NO: 58) QDMPGYFD (SEQ ID NO: 67) TNQNVDYSGNTF (SEQ ID NO: 77) LAS (SEQ ID NO: 83) SRNLPY (SEQ ID NO: 88) Honegger VSGFSLSIYG (SEQ ID NO: 53) IWSGGRKDYSLSLKSR (SEQ ID NO: 62) SQDMPGYFD (SEQ ID NO: 68) TNQNVDYSGNTF (SEQ ID NO: 77) LASNLASGIPER (SEQ ID NO: 85) SRNLPY (SEQ ID NO: 88) IMGT GFSLSIYG (SEQ ID NO: 54) IWSGGRK (SEQ ID NO: 60) ARSQDMPGYFDY (SEQ ID NO: 69) NQNVDYSGNTF (SEQ ID NO: 78) LAS (SEQ ID NO: 83) QQSRNLPYT (SEQ ID NO: 25) Kabat IYGVN (SEQ ID NO: 26) AIWSGGRKDYSLSLKS (SEQ ID NO: 63) SQDMPGYFDY (SEQ ID NO: 28) KTNQNVDYSGNTFMH (SEQ ID NO: 79) LASNLAS (SEQ ID NO: 24) QQSRNLPYT (SEQ ID NO: 25) ABC2067 Chothia GFSLSIY (SEQ ID NO: 52) SGG (SEQ ID NO: 58) QDMPGYFD (SEQ ID NO: 67) TNQNVDYSGNTF (SEQ ID NO: 77) LAS (SEQ ID NO: 83) SRNLPY (SEQ ID NO: 88) Honegger VSGFSLSIYG (SEQ ID NO: 53) IWSGGRKDYSLSLKSR (SEQ ID NO: 62) SQDMPGYFD (SEQ ID NO: 68) TNQNVDYSGNTF (SEQ ID NO: 77) LASNLASGIPER (SEQ ID NO: 85) SRNLPY (SEQ ID NO: 88) IMGT GFSLSIYG (SEQ ID NO: 54) IWSGGRK (SEQ ID NO: 60) ARSQDMPGYFDY (SEQ ID NO: 69) NQNVDYSGNTF (SEQ ID NO: 78) LAS (SEQ ID NO: 83) QQSRNLPYT (SEQ ID NO: 25) Kabat IYGVN (SEQ ID NO: 26) AIWSGGRKDYSLSLKS (SEQ ID NO: 63) SQDMPGYFDY (SEQ ID NO: 28) KTNQNVDYSGNTFMH (SEQ ID NO: 79) LASNLAS (SEQ ID NO: 24) QQSRNLPYT (SEQ ID NO: 25) ABC2097 Chothia GFSLSIY (SEQ ID NO: 52) SGG (SEQ ID NO: 58) QDMPGYFD (SEQ ID NO: 67) TNQNVDYSGNTF (SEQ ID NO: 77) LAS (SEQ ID NO: 83) SRNLPY (SEQ ID NO: 88) Honegger VSGFSLSIYG (SEQ ID NO: 53) IWSGGRKDYSLSLKSR (SEQ ID NO: 62) SQDMPGYFD (SEQ ID NO: 68) TNQNVDYSGNTF (SEQ ID NO: 77) LASNLASGIPER (SEQ ID NO: 85) SRNLPY (SEQ ID NO: 88) IMGT GFSLSIYG (SEQ ID NO: 54) IWSGGRK (SEQ ID NO: 60) ARSQDMPGYFDY (SEQ ID NO: 69) NQNVDYSGNTF (SEQ ID NO: 78) LAS (SEQ ID NO: 83) QQSRNLPYT (SEQ ID NO: 25) Kabat IYGVN (SEQ ID NO: 26) AIWSGGRKDYSLSLKS (SEQ ID NO: 63) SQDMPGYFDY (SEQ ID NO: 28) KTNQNVDYSGNTFMH (SEQ ID NO: 79) LASNLAS (SEQ ID NO: 24) QQSRNLPYT (SEQ ID NO: 25)

下 表 8中的序列繪示可用以產生本揭露的FAP結合劑（例如抗FAP抗體、雙特異性抗體、或其融合蛋白）及/或系統及組成物且執行本文所述方法的例示性VH及VL胺基酸序列。根據需要，可容易地從胺基酸序列推導出核苷酸序列。 表8 ：例示性VH 及VL 胺基酸序列 AB VH AB VL 9E3 嵌合體(ABC1139) QVQLKESGPGMVQPSRTLSLTCTVSGFSLSIYGVNWVRQPPGKGLEWIAAIWSGGRKDYNLSLKSRLIISGDTSKSQVLLTMNSLQSEDTAMYFCARSQDMPGYFDYWGQGVMVTVSS (SEQ ID NO: 35) 9E3 嵌合體(ABC1139) DIVLTQSPALAVSLGQRATISCKTNQNVDYNGNTFMHWYQQKPGQQPKLLLYLASNLASGIPDRFSGRGSGTDFTLTIDPVEADDTATYYCQQSRNLPYTFGAGTKLEIK (SEQ ID NO: 36) ABC1930 、ABC1931 、ABC1947 、ABC2066 、ABC2067 、ABC2097 EVQLQESGPGLVKPSETLSLTCTVSGFSLSIYGVNWVRQPPGKGLEWIAAIWSGGRKDYSLSLKSRLTISGDTSKNQVSLKLSSVTAADTAVYYCARSQDMPGYFDYWGQGTLVTVSS (SEQ ID NO: 37) ABC1930 、ABC1931 、ABC1947 、ABC2066 、ABC2067 、ABC2097 SIVLTQPPSVSVAPGQTARITCKTNQNVDYSGNTFMHWYQQKPGQQPVLLLYLASNLASGIPERFSGSGSGTTFTLTISRVEAGDEADYYCQQSRNLPYTFGTGTKVTVL (SEQ ID NO: 38) ABC1773 QVTLRESGPALVKPTQTLTLTCTFSGFSLSTAGMSVGWIRQPPGKALEWLADIWWDDKKHYNPSLKDRLTISKDTSKNQVVLKVTNMDPADTATYYCARDMIFNFYFDVWGQGTTVTVSS (SEQ ID NO: 41) ABC1773 DIQMTQSPSTLSASVGDRVTITCSASSRVGYMHWYQQKPGKAPKLLIYDTSKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCFQGSGYPFTFGGGTKVEIK (SEQ ID NO: 42) The sequences illustrated in Table 8 below are exemplary VH and VL amino acid sequences that can be used to generate the FAP binding agents (e.g., anti-FAP antibodies, bispecific antibodies, or fusion proteins thereof) and/or systems and compositions disclosed herein and to perform the methods described herein. The nucleotide sequences can be readily deduced from the amino acid sequences if needed. Table 8 : Exemplary VH and VL Amino Acid Sequences AB VH AB VL 9E3 chimera (ABC1139) QVQLKESGPGMVQPSRTLSLTCTVSGFSLSIYGVNWVRQPPGKGLEWIAAIWSGGRKDYNLSLKSRLIISGDTSKSQVLLTMNSLQSEDTAMYFCARSQDMPGYFDYWGQGVMVTVSS (SEQ ID NO: 35) 9E3 chimera (ABC1139) DIVLTQSPALAVSLGQRATISCKTNQNVDYNGNTFMHWYQQKPGQQPKLLLYLASNLASGIPDRFSGRGSGTTDFTLTIDPVEADDTATYYCQQSRNLPYTFGAGTKLEIK (SEQ ID NO: 36) ABC1930 , ABC1931 , ABC1947 , ABC2066 , ABC2067 , ABC2097 EVQLQESGPGLVKPSETLSLTCTVSGFSLSIYGVNWVRQPPGKGLEWIAAIWSGGRKDYSLSLKSRLTISGDTSKNQVSLKLSSVTAADTAVYYCARSQDMPGYFDYWGQGTLVTVSS (SEQ ID NO: 37) ABC1930 , ABC1931 , ABC1947 , ABC2066 , ABC2067 , ABC2097 SIVLTQPPSVSVAPGQTARITCKTNQNVDYSGNTTFMHWYQQKPGQQPVLLLYLASNLASGIPERFSGSGSGTTFTLTISRVEAGDEADYYCQQSRNLPYTFGTGTKVTVL (SEQ ID NO: 38) ABC1773 QVTLRESGPALVKPTQTLTLTCTFSGFSLSTAGMSVGWIRQPPGKALEWLADIWWDDKKHYNPSLKDRLTISKDTSKNQVVLKVTNMDPADTATYYCARDMIFNFYFDVWGQGTTVTVSS (SEQ ID NO: 41) ABC1773 DIQMTQSPSTLSASSVGDRVTITCSASSRVGYMHWYQQKPGKAPKLLIYDTSKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCFQGSGYPFTFGGGTKVEIK (SEQ ID NO: 42)

下 表 9中的序列繪示可用以產生本揭露的FAP結合劑（例如抗FAP抗體、雙特異性抗體、或其融合蛋白）及/或系統及組成物且執行本文所述方法的例示性HC及LC胺基酸序列。根據需要，可容易地從胺基酸序列推導出核苷酸序列。 表9 ：例示性HC 及LC 胺基酸序列 AB HC LC 9E3 嵌合體(ABC1139) QVQLKESGPGMVQPSRTLSLTCTVSGFSLSIYGVNWVRQPPGKGLEWIAAIWSGGRKDYNLSLKSRLIISGDTSKSQVLLTMNSLQSEDTAMYFCARSQDMPGYFDYWGQGVMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 1) DIVLTQSPALAVSLGQRATISCKTNQNVDYNGNTFMHWYQQKPGQQPKLLLYLASNLASGIPDRFSGRGSGTDFTLTIDPVEADDTATYYCQQSRNLPYTFGAGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 2) ABC1931 EVQLQESGPGLVKPSETLSLTCTVSGFSLSIYGVNWVRQPPGKGLEWIAAIWSGGRKDYSLSLKSRLTISGDTSKNQVSLKLSSVTAADTAVYYCARSQDMPGYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 4) SIVLTQPPSVSVAPGQTARITCKTNQNVDYSGNTFMHWYQQKPGQQPVLLLYLASNLASGIPERFSGSGSGTTFTLTISRVEAGDEADYYCQQSRNLPYTFGTGTKVTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 3) AB HC1 HC2 LC ABC1930 EVQLQESGPGLVKPSETLSLTCTVSGFSLSIYGVNWVRQPPGKGLEWIAAIWSGGRKDYSLSLKSRLTISGDTSKNQVSLKLSSVTAADTAVYYCARSQDMPGYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLGGMGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWQDSSFLGGVVHLEAGEKVVVRVLDERLVRLLDGTRSYFGAFMV (SEQ ID NO: 5) EVQLQESGPGLVKPSETLSLTCTVSGFSLSIYGVNWVRQPPGKGLEWIAAIWSGGRKDYSLSLKSRLTISGDTSKNQVSLKLSSVTAADTAVYYCARSQDMPGYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLGGMGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWQDSSFLGGVVHLEAGEKVVVRVLDERLVRLLDGTRSYFGAFMVGGGGSGGGGSEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLGGMGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWQDSSFLGGVVHLEAGEKVVVRVLDERLVRLLDGTRSYFGAFMV (SEQ ID NO: 6) SIVLTQPPSVSVAPGQTARITCKTNQNVDYSGNTFMHWYQQKPGQQPVLLLYLASNLASGIPERFSGSGSGTTFTLTISRVEAGDEADYYCQQSRNLPYTFGTGTKVTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 3) ABC1773 QVTLRESGPALVKPTQTLTLTCTFSGFSLSTAGMSVGWIRQPPGKALEWLADIWWDDKKHYNPSLKDRLTISKDTSKNQVVLKVTNMDPADTATYYCARDMIFNFYFDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLGGMGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWQDSSFLGGVVHLEAGEKVVVRVLDERLVRLLDGTRSYFGAFMV* (SEQ ID NO: 8) QVTLRESGPALVKPTQTLTLTCTFSGFSLSTAGMSVGWIRQPPGKALEWLADIWWDDKKHYNPSLKDRLTISKDTSKNQVVLKVTNMDPADTATYYCARDMIFNFYFDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLGGMGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWQDSSFLGGVVHLEAGEKVVVRVLDERLVRLLDGTRSYFGAFMVGGGGSGGGGSEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLGGMGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWQDSSFLGGVVHLEAGEKVVVRVLDERLVRLLDGTRSYFGAFMV* (SEQ ID NO: 9) DIQMTQSPSTLSASVGDRVTITCSASSRVGYMHWYQQKPGKAPKLLIYDTSKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCFQGSGYPFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* (SEQ ID NO: 7) ABC1947 EVQLQESGPGLVKPSETLSLTCTVSGFSLSIYGVNWVRQPPGKGLEWIAAIWSGGRKDYSLSLKSRLTISGDTSKNQVSLKLSSVTAADTAVYYCARSQDMPGYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLGGVGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEKVVVRVLDERLVRLRDGTRSYFGAFMV (SEQ ID NO: 10) EVQLQESGPGLVKPSETLSLTCTVSGFSLSIYGVNWVRQPPGKGLEWIAAIWSGGRKDYSLSLKSRLTISGDTSKNQVSLKLSSVTAADTAVYYCARSQDMPGYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLGGVGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEKVVVRVLDERLVRLRDGTRSYFGAFMVGGGGSGGGGSEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLGGVGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEKVVVRVLDERLVRLRDGTRSYFGAFMV (SEQ ID NO: 11) SIVLTQPPSVSVAPGQTARITCKTNQNVDYSGNTFMHWYQQKPGQQPVLLLYLASNLASGIPERFSGSGSGTTFTLTISRVEAGDEADYYCQQSRNLPYTFGTGTKVTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 3) ABC2066 EVQLQESGPGLVKPSETLSLTCTVSGFSLSIYGVNWVRQPPGKGLEWIAAIWSGGRKDYSLSLKSRLTISGDTSKNQVSLKLSSVTAADTAVYYCARSQDMPGYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK* (SEQ ID NO: 13) RRSHEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKTGYYYIYSKVQLGGVGCPLGLAGTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEKVVVRVLGKRLVRLRDGTRSYFGAFMVGRRSHEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKTGYYYIYSKVQLGGVGCPLGLAGTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEKVVVRVLGKRLVRLRDGTRSYFGAFMVGRRSHEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKTGYYYIYSKVQLGGVGCPLGLAGTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEKVVVRVLGKRLVRLRDGTRSYFGAFMVGGGGSGGGGSGGGGSGGGGTGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK* (SEQ ID NO: 12) SIVLTQPPSVSVAPGQTARITCKTNQNVDYSGNTFMHWYQQKPGQQPVLLLYLASNLASGIPERFSGSGSGTTFTLTISRVEAGDEADYYCQQSRNLPYTFGTGTKVTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 3) ABC2067 RRSHEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKTGYYYIYSKVQLGGVGCPLGLAGTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEKVVVRVLGKRLVRLRDGTRSYFGAFMVGRRSHEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKTGYYYIYSKVQLGGVGCPLGLAGTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEKVVVRVLGKRLVRLRDGTRSYFGAFMVGRRSHEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKTGYYYIYSKVQLGGVGCPLGLAGTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEKVVVRVLGKRLVRLRDGTRSYFGAFMVGGGGSGGGGSGGGGSGGGGTCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK* (SEQ ID NO: 14) EVQLQESGPGLVKPSETLSLTCTVSGFSLSIYGVNWVRQPPGKGLEWIAAIWSGGRKDYSLSLKSRLTISGDTSKNQVSLKLSSVTAADTAVYYCARSQDMPGYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK* (SEQ ID NO: 13) SIVLTQPPSVSVAPGQTARITCKTNQNVDYSGNTFMHWYQQKPGQQPVLLLYLASNLASGIPERFSGSGSGTTFTLTISRVEAGDEADYYCQQSRNLPYTFGTGTKVTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 3) ABC2097 EVQLQESGPGLVKPSETLSLTCTVSGFSLSIYGVNWVRQPPGKGLEWIAAIWSGGRKDYSLSLKSRLTISGDTSKNQVSLKLSSVTAADTAVYYCARSQDMPGYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSLSPGLPAAHLIGAPLKGQGLGWETTKEQAFLTSGTQFSDAEGLALPQDGLYYLYCLVGYRGRAPPGGGDPQGRSVTLRSSLYRAGGAYGPGTPELLLEGAETVTPVLDPARRQGYGPLWYTSVGFGGLVQLRRGERVYVNISHPDMVDFARGKTFFGAVMVG* (SEQ ID NO: 15) EVQLQESGPGLVKPSETLSLTCTVSGFSLSIYGVNWVRQPPGKGLEWIAAIWSGGRKDYSLSLKSRLTISGDTSKNQVSLKLSSVTAADTAVYYCARSQDMPGYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSAHSTLKPAAHLIGDPSKQNSLLWRANTDRAFLQDGFSLSNNSLLVPTSGIYFVYSQVVFSGKAYSPKATSSPLYLAHEVQLFSSQYPFHVPLLSSQKMVYPGLQEPWLHSMYHGAAFQLTQGDQLSTHTDGIPHLVLSPSTVFFGAFALLSPGLPAAHLIGAPLKGQGLGWETTKEQAFLTSGTQFSDAEGLALPQDGLYYLYCLVGYRGRAPPGGGDPQGRSVTLRSSLYRAGGAYGPGTPELLLEGAETVTPVLDPARRQGYGPLWYTSVGFGGLVQLRRGERVYVNISHPDMVDFARGKTFFGAVMVG* (SEQ ID NO: 16) SIVLTQPPSVSVAPGQTARITCKTNQNVDYSGNTFMHWYQQKPGQQPVLLLYLASNLASGIPERFSGSGSGTTFTLTISRVEAGDEADYYCQQSRNLPYTFGTGTKVTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 3) The sequences illustrated in Table 9 below are exemplary HC and LC amino acid sequences that can be used to generate the FAP binding agents (e.g., anti-FAP antibodies, bispecific antibodies, or fusion proteins thereof) and/or systems and compositions disclosed herein and to perform the methods described herein. Nucleotide sequences can be readily deduced from amino acid sequences if needed. Table 9 : Exemplary HC and LC Amino Acid Sequences AB HC LC 9E3 chimera (ABC1139) QVQLKESGPGMVQPSRTLSLTCTVSGFSLSIYGVNWVRQPPGKGLEWIAAIWSGGRKDYNLSLKSRLIISGDTSKSQVLLTMNSLQSEDTAMYFCARSQDMPGYFDYWGQGV MVTVSSASTKGPSVFPLAPSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 1) DIVLTQSPALAVSLGQRATISCKTNQNVDYNGNTFMHWYQQKPGQQPKLLLYLASNLASGIPDRFSGRGSGTDFTLTIDPVEADDTATYYCQQSRNLPYTFGAGTKLE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 2) ABC1931 EVQLQESGPGLVKPSETLSLTCTVSGFSLSIYGVNWVRQPPGKGLEWIAAIWSGGRKDYSLSLKSRLTISGDTSKNQVSLKLSSVTAADTAVYYCARSQDMPGYFDYWGQG TLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGP PCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 4) SIVLTQPPSVSVVAPGQTARITCKTNQNVDYSGNTTFMHWYQQKPGQQPVLLLYLASNLASGIPERFSGSGSGTTFTLTISRVEAGDEADYYCQQSRNLPYTFGTGTKVT VLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 3) AB HC1 HC2 LC ABC1930 EVQLQESGPGLVKPSETLSLTCTVSGFSLSIYGVNWVRQPPGKGLEWIAAIWSGGRKDYSLSLKSRLTISGDTSKNQVSLKLSSVTAADTAVYYCARSQDMPGYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGG GSEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLGGMGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWQDSSFLGGVVHLEAGEKVVVRVLDERLVRLLDGTRSYFGAFMV (SEQ ID NO: 5) EVQLQESGPGLVKPSETLSLTCTVSGFSLSIYGVNWVRQPPGKGLEWIAAIWSGGRKDYSLSLKSRLTISGDTSKNQVSLKLSSVTAADTAVYYCARSQDMPGYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS SSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAK TKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLGKGGGGSGGGGSEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLGGMGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWQDSSFLGGVVH LEAGEKVVVRVLDERLVRLLDGTRSYFGAFMVGGGGSGGGGSEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLGGMGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWQDSSFLGGVVHLEAGEKVVVRVLDERLVRLLDGTRSYFGAFMV (SEQ ID NO: 6) SIVLTQPPSVSVVAPGQTARITCKTNQNVDYSGNTTFMHWYQQKPGQQPVLLLYLASNLASGIPERFSGSGSGTTFTLTISRVEAGDEADYYCQQSRNLPYTFGTGTKVT VLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 3) ABC1773 * (SEQ ID NO: 8) * (SEQ ID NO: 9) DIQMTQSPSTLSASVGDRVTITCSASSRVGYMHWYQQKPGKAPKLLIYDTSKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCFQGSGYPFTFGGGTKVEIKR TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* (SEQ ID NO: 7) ABC1947 EVQLQESGPGLVKPSETLSLTCTVSGFSLSIYGVNWVRQPPGKGLEWIAAIWSGGRKDYSLSLKSRLTISGDTSKNQVSLKLSSVTAADTAVYYCARSQDMPGYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGG GSEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLGGVGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEKVVVRVLDERLVRLRDGTRSYFGAFMV (SEQ ID NO: 10) EVQLQESGPGLVKPSETLSLTCTVSGFSLSIYGVNWVRQPPGKGLEWIAAIWSGGRKDYSLSLKSRLTISGDTSKNQVSLKLSSVTAADTAVYYCARSQDMPGYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS SSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAK TKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLGKGGGGSGGGGSEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLGGVGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVH LEAGEKVVVRVLDERLVRLRDGTRSYFGAFMVGGGGSGGGGSEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLGGVGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEKVVVRVLDERLVRLRDGTRSYFGAFMV (SEQ ID NO: 11) SIVLTQPPSVSVVAPGQTARITCKTNQNVDYSGNTTFMHWYQQKPGQQPVLLLYLASNLASGIPERFSGSGSGTTFTLTISRVEAGDEADYYCQQSRNLPYTFGTGTKVT VLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 3) ABC2066 EVQLQESGPGLVKPSETLSLTCTVSGFSLSIYGVNWVRQPPGKGLEWIAAIWSGGRKDYSLSLKSRLTISGDTSKNQVSLKLSSVTAADTAVYYCARSQDMPGYFDYWGQG TLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPP CPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTI SKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK* (SEQ ID NO: 13) * (SEQ ID NO: 12) SIVLTQPPSVSVVAPGQTARITCKTNQNVDYSGNTTFMHWYQQKPGQQPVLLLYLASNLASGIPERFSGSGSGTTFTLTISRVEAGDEADYYCQQSRNLPYTFGTGTKVT VLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 3) ABC2067 * (SEQ ID NO: 14) EVQLQESGPGLVKPSETLSLTCTVSGFSLSIYGVNWVRQPPGKGLEWIAAIWSGGRKDYSLSLKSRLTISGDTSKNQVSLKLSSVTAADTAVYYCARSQDMPGYFDYWGQG TLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPP CPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTI SKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK* (SEQ ID NO: 13) SIVLTQPPSVSVVAPGQTARITCKTNQNVDYSGNTTFMHWYQQKPGQQPVLLLYLASNLASGIPERFSGSGSGTTFTLTISRVEAGDEADYYCQQSRNLPYTFGTGTKVT VLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 3) ABC2097 * (SEQ ID NO: 15) * (SEQ ID NO: 16) SIVLTQPPSVSVVAPGQTARITCKTNQNVDYSGNTTFMHWYQQKPGQQPVLLLYLASNLASGIPERFSGSGSGTTFTLTISRVEAGDEADYYCQQSRNLPYTFGTGTKVT VLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 3)

下 表 10中的序列繪示可用以產生本揭露的FAP結合劑（例如抗FAP抗體、雙特異性抗體、或其融合蛋白）及/或系統及組成物且執行本文所述方法的例示性腫瘤相關細胞受體配體胺基酸序列。根據需要，可容易地從胺基酸序列推導出核苷酸序列。 表10 ：例示性胺基酸序列 SEQ ID NO: 描述 序列 17 LIGHT1突變體 EVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLGGMGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWQDSSFLGGVVHLEAGEKVVVRVLDERLVRLLDGTRSYFGAFMV 18 野生型LIGHT EVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLGGVGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEKVVVRVLDERLVRLRDGTRSYFGAFMV 39 淋巴毒素B突變體 LSPGLPAAHLIGAPLKGQGLGWETTKEQAFLTSGTQFSDAEGLALPQDGLYYLYCLVGYRGRAPPGGGDPQGRSVTLRSSLYRAGGAYGPGTPELLLEGAETVTPVLDPARRQGYGPLWYTSVGFGGLVQLRRGERVYVNISHPDMVDFARGKTFFGAVMVG 40 淋巴毒素A突變體 AHSTLKPAAHLIGDPSKQNSLLWRANTDRAFLQDGFSLSNNSLLVPTSGIYFVYSQVVFSGKAYSPKATSSPLYLAHEVQLFSSQYPFHVPLLSSQKMVYPGLQEPWLHSMYHGAAFQLTQGDQLSTHTDGIPHLVLSPSTVFFGAFAL 實例 The sequences illustrated in Table 10 below are exemplary tumor-associated cell receptor ligand amino acid sequences that can be used to generate the FAP binding agents (e.g., anti-FAP antibodies, bispecific antibodies, or fusion proteins thereof) and/or systems and compositions disclosed herein and to perform the methods described herein. Nucleotide sequences can be readily deduced from the amino acid sequences if needed. Table 10 : Exemplary Amino Acid Sequences SEQ ID NO: describe sequence 17 LIGHT1 Mutant EVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLGGMGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWQDSSFLGGVVHLEAGEKVVVRVLDERLVRLLDGTRSYFGAFMV 18 Wild type LIGHT EVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLGGVGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEKVVVRVLDERLVRLRDGTRSYFGAFMV 39 Lymphotoxin B mutant LSPGLPAAHLIGAPLKGQGLGWETTKEQAFLTSGTQFSDAEGLALPQDGLYYLYCLVGYRGRAPPGGGDPQGRSVTLRSSLYRAGGAYGPGTPELLLEGAETVTPVLDPARRQGYGPLWYTSVGFGGLVQLRRGERVYVNISHPDMVDFARGKTFFGAVMVG 40 Lymphotoxin A mutant AHSTLKPAAHLIGDPSKQNSLLWRANTDRAFLQDGFSLSNSLVPTSGIYFVYSQVVFSGKAYSPKATSSPLYLAHEVQLFSSQYPFHVPLLSSQKMVYPGLQEPWLHSMYHGAAFQLTQGDQLSTHTDGIPHLVLSPSTVFFGAFAL Example

以下實例進一步描述本發明，其不限制申請專利範圍中所描述之本發明之範疇。實例中使用的試劑係市售的。The following examples further illustrate the invention, but do not limit the scope of the invention as described in the patent application. The reagents used in the examples are commercially available.

通常，與本文所描述的細胞及組織培養、病理學、腫瘤學、分子生物學、免疫學、微生物學、遺傳學、及蛋白質及核酸化學及雜交相關使用的命名法及技術係所屬技術領域中熟知及常用的那些者。除非另有說明，否則本揭露的方法及技術大致上根據所屬技術領域中熟知的習知方法且如本說明書通篇中所引述及討論之各種一般且更具體參考文獻中所描述般執行。參見例如Green and Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)；Therapeutic Monoclonal Antibodies: From Bench to Clinic, Zhiqiang An (Editor), Wiley, (2009)；及Antibody Engineering, 2nd Ed., Vols 1 and 2, Ontermann and Dubel, eds., Springer-Verlag, Heidelberg (2010)。 實例1 ：抗FAP 抗體的產生1.1.融合瘤產生及篩選 Generally, the nomenclature and techniques used in relation to cell and tissue culture, pathology, oncology, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the relevant art. Unless otherwise stated, the methods and techniques disclosed herein are generally performed in accordance with well-known practices in the relevant art and as described in the various general and more specific references cited and discussed throughout this specification. See, for example, Green and Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012); Therapeutic Monoclonal Antibodies: From Bench to Clinic, Zhiqiang An (Editor), Wiley, (2009); and Antibody Engineering, 2nd Ed., Vols 1 and 2, Ontermann and Dubel, eds., Springer-Verlag, Heidelberg (2010). Example 1 : Generation of anti-FAP antibodies 1.1. Generation and screening of fusion tumors

為了產生針對FAP的抗體，用FAP過度表現細胞對Sprague-Dawley大鼠進行免疫。為了監測免疫反應，一般在免疫4至6週後，由FACS篩選滴定血清與多種FAP過度表現細胞系的結合。選擇具有足夠滴度的抗FAP IgG的動物進行最終免疫。To generate antibodies against FAP, Sprague-Dawley rats were immunized with FAP-overexpressing cells. To monitor the immune response, serum was titrated with various FAP-overexpressing cell lines using FACS screening, typically 4 to 6 weeks after immunization. Animals with sufficient titers of anti-FAP IgG were selected for final immunization.

為了產生融合瘤，收集、單離來自淋巴器官（諸如脾臟及淋巴結）的細胞，且與SP2/0骨髓瘤細胞融合。將所得細胞接種在補充有HAT (Sigma H0262-10VL)的培養基E (StemCell Technologies)中的平底96孔盤中以選擇融合瘤。培養一週之後，收集融合瘤上清液且以流式細胞術篩選過度表現FAP的細胞。隨後對所選擇之融合瘤進行次選殖且進一步篩選以識別抗FAP單株融合瘤。 1.2.表現載體的產生 To generate fusion tumors, cells from lymphoid organs (such as the spleen and lymph nodes) were collected, isolated, and fused with SP2/0 myeloma cells. The resulting cells were seeded in flat-bottomed 96-well plates in StemCell Technologies medium supplemented with HAT (Sigma H0262-10VL) to select fusion tumors. After one week of culture, the supernatant from the fusion tumors was collected, and cells overexpressing FAP were screened by flow cytometry. The selected fusion tumors were then secondary-selected and further screened to identify anti-FAP monoclonal fusion tumors. 1.2. Generation of Expression Vectors

為了獲得重鏈及輕鏈可變域的序列，使用Dynabeads mRNA Direct Micro Purification套組(ThermoFisher #61021)純化來自融合瘤的mRNA，且使用模板轉換寡核苷酸合成及擴增cDNA (Pinto et al., Anal. Biochem. 397, 2010)。隨後對重鏈及輕鏈的可變域片段進行擴增、純化、及定序。To obtain the sequences of the heavy and light chain variable domains, mRNA from fusion tumors was purified using the Dynabeads mRNA Direct Micro Purification kit (ThermoFisher #61021), and cDNA was synthesized and amplified using template-conversion oligonucleotides (Pinto et al., Anal. Biochem. 397, 2010). Subsequently, the variable domain fragments of the heavy and light chains were amplified, purified, and sequenced.

為了產生表現載體，合成重鏈及輕鏈DNA序列的可變區，且將其與人類IgG1恆定重鏈或預插入pCI載體(Promega#E1841)的人類IgG1 κ恆定輕鏈進行框內次選殖。9E3嵌合體(ABC1139)的CDR及可變區係如下所顯示。To generate the expression vector, variable regions of the heavy and light chain DNA sequences were synthesized and then in-frame secondary colonized with either the human IgG1 constant heavy chain or the human IgG1 κ constant light chain pre-inserted into the pCI vector (Promega#E1841). The CDR and variable region structure of the 9E3 chimera (ABC1139) are shown below.

重鏈(AP1333)： QVQLKESGPGMVQPSRTLSLTCTVSGFSLS IYGVNWVRQPPGKGLEWIA AIWSGGRKDYNLSLKSRLIISGDTSKSQVLLTMNSLQSEDTAMYFCAR SQDMPGYFDYWGQGVMVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 1) 輕鏈(AP1334)： DIVLTQSPALAVSLGQRATISC KTNQNVDYNGNTFMHWYQQKPGQQPKLLLY LASNLASGIPDRFSGRGSGTDFTLTIDPVEADDTATYYC QQSRNLPYTFGAGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 2) 將CDR加底線並使用Kabat系統定義。可變區係以粗體顯示。 1.3.抗體的人源化 Heavy chain (AP1333): QVQLKESGPGMVQPSRTLSLTCTVSGFSLS IYGVN WVRQPPGKGLEWIA AIWSGGRKDYNLSLKS RLIISGDTKSSQVLLTMNSLQSEDTAMYFCAR SQDMPGYFDY WGQGVMVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 1) Light chain (AP1334): DIVLTQSPALAVSLGQRATISC KTNQNVDYNGNTFMH WYQQKPGQQPKLLLY LASNLAS GIPDRFSGRGSGTDFTLTIDPVEADDTATYYC QQSRNLPYT FGAGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 2) Underline the CDR and define it using the Kabat system. Variable regions are shown in bold. 1.3. Humanization of Antibodies

為了使抗體人源化，將輕鏈及重鏈的CDR殘基接枝到具有高度同源性的人類架構上。為了恢復親和力，藉由在具體位置將人類架構殘基改變為其等小鼠對應物來設計一些突變。移除轉譯後修飾(PTM)。To humanize the antibody, CDR residues from both the light and heavy chains were grafted onto highly homologous human structures. To restore affinity, mutations were designed by altering the human structural residues at specific sites to their corresponding mouse counterparts. Post-translational modifications (PTMs) were removed.

人源化抗體ABC1931的CDR及可變區係如下所顯示。 輕鏈(AP2463)： SIVLTQPPSVSVAPGQTARITC KTNQNVDYSGNTFMHWYQQKPGQQPVLLLY LASNLASGIPERFSGSGSGTTFTLTISRVEAGDEADYYC QQSRNLPYTFGTGTKVTVL GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 3) 重鏈(AP1992)： EVQLQESGPGLVKPSETLSLTCTVSGFSLS IYGVNWVRQPPGKGLEWIA AIWSGGRKDYSLSLKSRLTISGDTSKNQVSLKLSSVTAADTAVYYCAR SQDMPGYFDYWGQGTLVTVSS ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 4) 將CDR加底線並使用Kabat系統定義。可變區係以粗體顯示。 1.4抗體的產生 The CDR and variable regions of the humanized antibody ABC1931 are shown below. Light chain (AP2463): SIVLTQPPSVSVVAPGQTARITC KTNQNVDYSGNTFMH WYQQKPGQQPVLLLY LASNLAS GIPERFSGSGSGTTFTLTISRVEAGDEADYYC QQSRNLPYT FGTGTKVTVL GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 3) Heavy chain (AP1992): EVQLQESGPGLVKPSETLSLTCTVSGFSLS IYGVN WVRQPPGKGLEWIA AIWSGGRKDYSLSLKS RLTISGDTSKNQVSLKLSSVTAADTAVYYCAR SQDMPGYFDY WGQGTLVTVSS ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 4) Underline the CDR and define it using the Kabat system. Variable regions are shown in bold. 1.4 Antibody Production

將質體共轉染至Expi293F細胞(ThermoFisher A14527)中來產生抗體。具體而言，將30 µg總DNA稀釋到Opti-MEM培養基(Life Technologies)中，且接著與Expifectamine 293轉染試劑(Gibco #A14525)在同一培養基中培養20 min。接著將混合物添加到在37℃及8% CO 2下以250萬個細胞/ml的密度在懸浮液中生長的30 ml Expi293F細胞中。 Antibodies were generated by co-transfecting plasmids into Expi293F cells (ThermoFisher A14527). Specifically, 30 µg of total DNA was diluted in Opti-MEM medium (Life Technologies) and then incubated for 20 min in the same medium as Expifectamine 293 transfection reagent (Gibco #A14525). The mixture was then added to 30 ml of Expi293F cells grown in suspension at 37°C and 8% CO2 at a density of 2.5 million cells/ml.

轉染之後，細胞在懸浮液中生長約六天，且接著藉由在4℃下以7,000 rpm離心20分鐘來進行收取。將上清液通過0.22 µm過濾器過濾，且使用蛋白質A樹脂執行親和純化。將蛋白質用洗提緩衝液（1 M甘胺酸pH 3.0、10%甘油）洗提並用1M Tris pH 7.5中和至pH 6.0。在AKTA上執行粒徑排阻層析法(size exclusion chromatography, SEC)以研磨產物，且用NanoDrop在280 nm處測量抗體濃度。在還原(reducing, R)及非還原(non-reducing, NR)條件下，藉由在4至20% Tris-甘胺酸SDS-PAGE凝膠(Bio-Rad)上進行電泳來分析抗體。 1.5.產生過度表現受體或目標的穩定細胞系 Following transfection, cells were grown in suspension for approximately six days and then harvested by centrifugation at 7,000 rpm for 20 minutes at 4°C. The supernatant was filtered through a 0.22 µm filter and purified using protein A resin for affinity. The protein was eluted with an elution buffer (1 M glycine, pH 3.0, 10% glycerol) and neutralized to pH 6.0 with 1 M Tris at pH 7.5. Size exclusion chromatography (SEC) was performed on an AKTA to grind the products, and antibody concentrations were measured at 280 nm using NanoDrop. Antibodies were analyzed by electrophoresis on 4–20% Tris-glycine SDS-PAGE gels (Bio-Rad) under reducing (R) and non-reducing (NR) conditions. 1.5. Stable cell lines that produce overexpressed receptors or targets

藉由使用lipofectamine 3000按照標準規程將全長人類、食蟹獼猴、或小鼠FAP（表1）轉染到HEK-293T細胞中來產生293T-FAP細胞系。三天之後，將細胞用潮黴素(Millipore Sigma)處理14天以產生穩定細胞系。用類似的程序產生293T-HVEM細胞系及293T-LTβR細胞系。 表1 ：FAP 、HVEM 、LTΒR 蛋白及序列來源 蛋白質名稱 序列來源 人類FAP Uniprot Q12884 食蟹獼猴FAP NCBI XP_005573377.1 小鼠FAP Uniprot P97321 人類HVEM Uniprot Q92956 食蟹獼猴HVEM NCBI XP_005545061.1 小鼠HVEM Uniprot Q80WM9 人類LTβR Uniprot P36941 食蟹獼猴LTβR Uniprot A0A2K5VGQ6 小鼠LTβR Uniprot P50284 由FACS進行細胞結合分析： 293T-FAP cell lines were generated by transfecting full-length human, cynomolgus monkey, or mouse FAP (Table 1) into HEK-293T cells using lipofectamine 3000 according to standard procedures. Three days later, the cells were treated with hygroscopic fungicide (Millipore Sigma) for 14 days to generate stable cell lines. 293T-HVEM and 293T-LTβR cell lines were generated using a similar procedure. Table 1 : FAP , HVEM , and LTβR protein and sequence sources. Protein name Sequence Source Human FAP Uniprot Q12884 Crab-eating macaques FAP NCBI XP_005573377.1 mouse FAP Uniprot P97321 Human HVEM Uniprot Q92956 Crab-eating macaques HVEM NCBI XP_005545061.1 mouse HVEM Uniprot Q80WM9 Human LTβR Uniprot P36941 Crab-eating macaque LTβR Uniprot A0A2K5VGQ6 mouse LTβR Uniprot P50284 Cell binding analysis using FACS:

使用穩定轉染的表現FAP之HEK-293T細胞評估抗FAP單株抗體(mAb)之結合活性。將細胞與連續稀釋的抗FAP單株抗體在冰上培養30分鐘，流式細胞術緩衝液(PBS, 0.5% BSA, 2 mM EDTA)中的最大濃度為50 nM。接著將細胞用PBS洗滌三次，且將結合的mAb用FITC接合的小鼠抗人類IgG抗體在冰上染色15分鐘。將細胞進一步用PBS洗滌三次，且使用Cytek Aurora細胞儀(Cytek)測量螢光，且使用FlowJo及GraphPad Prism 9.0軟體進行分析以判定EC 50。結果係彙總於表2中。 表2 ：抗FAP mAb 與表現FAP 之293T 細胞結合的EC50 EC 50(nM) 9E3嵌合體mAb ABC1931 （人源化9E3） hFAP 1.001 3.7 cynoFAP 0.616 1.4 mFAP 0.470 0.84 人類FAP、食蟹獼猴FAP、及小鼠FAP分別縮寫為hFAP、cynoFAP、及mFAP。 The binding activity of anti-FAP monoclonal antibodies (mAbs) was assessed using stably transfected FAP-expressing HEK-293T cells. Cells were incubated on ice for 30 min with serially diluted anti-FAP monoclonal antibodies at a maximum concentration of 50 nM in flow cytometry buffer (PBS, 0.5% BSA, 2 mM EDTA). Cells were then washed three times with PBS, and the bound mAbs were stained on ice for 15 min with FITC-conjugated mouse anti-human IgG antibodies. Cells were further washed three times with PBS, and fluorescence was measured using a Cytek Aurora cytometer (Cytek). Analysis was performed using FlowJo and GraphPad Prism 9.0 software to determine EC50 . The results are summarized in Table 2. Table 2 : EC50 binding of anti-FAP mAb to FAP-expressing 293T cells. EC 50 (nM) 9E3 chimera mAb ABC1931 (Humanized 9E3) hFAP 1.001 3.7 cynoFAP 0.616 1.4 mFAP 0.470 0.84 Human FAP, cynomolgus monkey FAP, and mouse FAP are abbreviated as hFAP, cynoFAP, and mFAP, respectively.

如圖1A至圖1C及表2所示，嵌合體抗體9E3與人類、食蟹獼猴、及小鼠FAP交叉反應，而人源化抗體ABC1931維持交叉反應性及親和力。 實例2 ：免疫細胞介素產生及選擇2.1.免疫細胞介素的產生 As shown in Figures 1A to 1C and Table 2, the chimeric antibody 9E3 cross-reacts with human, cynomolgus monkey, and mouse FAP antibodies, while the humanized antibody ABC1931 maintains both cross-reactivity and affinity. Example 2 : Production and Selection of Immuno-interstitial cells 2.1. Production of Immuno-interstitial cells

人源化9E3的可變域用以產生呈形式A及形式D兩者的免疫細胞介素。按照製造商的建議，藉由將具有不同重鏈(HC)質體比率的質體共轉染到Expi293F細胞(ThermoFisher#A14527)中來產生免疫細胞介素。Expi293F細胞在懸浮液培養物中生長。轉染後六天藉由離心收取上清液，且用0.22 µm過濾器進一步過濾，且以蛋白質A珠純化，且在非還原及還原條件下以SDS-PAGE凝膠進行分析。The variable domain of humanized 9E3 was used to generate immunocytokines in both form A and form D. Immunocytokines were generated by co-transfecting plasmids with different heavy chain (HC) plasmid ratios into Expi293F cells (ThermoFisher#A14527) as recommended by the manufacturer. Expi293F cells were grown in suspension culture. Six days post-transfection, the supernatant was collected by centrifugation, further filtered using a 0.22 µm filter, purified with protein A beads, and analyzed by SDS-PAGE gel under both reducing and non-reducing conditions.

經測試免疫細胞介素顯示於下 表 4 、表4.1、及 表 4.2中。 表4 ：經檢定抗體及重鏈/ 輕鏈 蛋白質概念ID 鏈 鏈代碼 蛋白質概念ID 鏈 鏈代碼 9E3嵌合體(ABC1139) HC AP1333 ABC1947 LC AP2463 HC1 AP1981 LC AP1334 HC2 AP2464 ABC1931 LC AP2463 ABC2066 LC AP2463 HC1 AP2586 HC AP1992 HC2 AP2584 ABC1930 LC AP2463 ABC2067 LC AP2463 HC1 AP1979 HC1 AP2585 HC2 AP2271 HC2 AP2586 ABC1773 LC AP672 ABC2097 LC AP2463 HC1 AP2267 HC1 AP2555 HC2 AP2269 HC2 AP2556 表4.1 ：經檢定抗體及Kabat CDR 蛋白質概念ID 鏈 CDR1 CDR2 CDR3 9E3嵌合體(ABC1139) HC IYGVN (SEQ ID NO: 26) AIWSGGRKDYNLSLKS (SEQ ID NO: 61) SQDMPGYFDY (SEQ ID NO: 28) LC KTNQNVDYNGNTFMH (SEQ ID NO: 76) LASNLAS (SEQ ID NO: 24) QQSRNLPYT (SEQ ID NO: 25) ABC1931 LC KTNQNVDYSGNTFMH (SEQ ID NO: 79) LASNLAS (SEQ ID NO: 24) QQSRNLPYT (SEQ ID NO: 25) HC IYGVN (SEQ ID NO: 26) AIWSGGRKDYSLSLKS (SEQ ID NO: 63) SQDMPGYFDY (SEQ ID NO: 28) ABC1930 LC KTNQNVDYSGNTFMH (SEQ ID NO: 79) LASNLAS (SEQ ID NO: 24) QQSRNLPYT (SEQ ID NO: 25) HC1 IYGVN (SEQ ID NO: 26) AIWSGGRKDYSLSLKS (SEQ ID NO: 63) SQDMPGYFDY (SEQ ID NO: 28) HC2 IYGVN (SEQ ID NO: 26) AIWSGGRKDYSLSLKS (SEQ ID NO: 63) SQDMPGYFDY (SEQ ID NO: 28) ABC1773 LC SASSRVGYMH (SEQ ID NO: 29) DTSKLAS (SEQ ID NO: 30) FQGSGYPFT (SEQ ID NO: 31) HC1 TAGMSVG (SEQ ID NO: 32) DIWWDDKKHYNPSLKD (SEQ ID NO: 33) DMIFNFYFDV (SEQ ID NO: 34) HC2 TAGMSVG (SEQ ID NO: 32) DIWWDDKKHYNPSLKD (SEQ ID NO: 33) DMIFNFYFDV (SEQ ID NO: 34) ABC1947 LC KTNQNVDYSGNTFMH (SEQ ID NO: 79) LASNLAS (SEQ ID NO: 24) QQSRNLPYT (SEQ ID NO: 25) HC1 IYGVN (SEQ ID NO: 26) AIWSGGRKDYSLSLKS (SEQ ID NO: 63) SQDMPGYFDY (SEQ ID NO: 28) HC2 IYGVN (SEQ ID NO: 26) AIWSGGRKDYSLSLKS (SEQ ID NO: 63) SQDMPGYFDY (SEQ ID NO: 28) ABC2066 LC KTNQNVDYSGNTFMH (SEQ ID NO: 79) LASNLAS (SEQ ID NO: 24) QQSRNLPYT (SEQ ID NO: 25) HC1 / / / HC2 IYGVN (SEQ ID NO: 26) AIWSGGRKDYSLSLKS (SEQ ID NO: 63) SQDMPGYFDY (SEQ ID NO: 28) ABC2067 LC KTNQNVDYSGNTFMH (SEQ ID NO: 79) LASNLAS (SEQ ID NO: 24) QQSRNLPYT (SEQ ID NO: 25) HC1 / / / HC2 IYGVN (SEQ ID NO: 26) AIWSGGRKDYSLSLKS (SEQ ID NO: 63) SQDMPGYFDY (SEQ ID NO: 28) ABC2097 LC KTNQNVDYSGNTFMH (SEQ ID NO: 79) LASNLAS (SEQ ID NO: 24) QQSRNLPYT (SEQ ID NO: 25) HC1 IYGVN (SEQ ID NO: 26) AIWSGGRKDYSLSLKS (SEQ ID NO: 63) SQDMPGYFDY (SEQ ID NO: 28) HC2 IYGVN (SEQ ID NO: 26) AIWSGGRKDYSLSLKS (SEQ ID NO: 63) SQDMPGYFDY (SEQ ID NO: 28) 表4.2 ：經檢定抗體及鏈胺基酸序列 鏈 代碼 單一代碼胺基酸序列 （可變區係以粗體顯示） AP1979 (SEQ ID NO: 5) EVQLQESGPGLVKPSETLSLTCTVSGFSLSIYGVNWVRQPPGKGLEWIAAIWSGGRKDYSLSLKSRLTISGDTSKNQVSLKLSSVTAADTAVYYCARSQDMPGYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLGGMGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWQDSSFLGGVVHLEAGEKVVVRVLDERLVRLLDGTRSYFGAFMV AP2271 (SEQ ID NO: 6) EVQLQESGPGLVKPSETLSLTCTVSGFSLSIYGVNWVRQPPGKGLEWIAAIWSGGRKDYSLSLKSRLTISGDTSKNQVSLKLSSVTAADTAVYYCARSQDMPGYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLGGMGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWQDSSFLGGVVHLEAGEKVVVRVLDERLVRLLDGTRSYFGAFMVGGGGSGGGGSEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLGGMGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWQDSSFLGGVVHLEAGEKVVVRVLDERLVRLLDGTRSYFGAFMV AP672 (SEQ ID NO: 7) DIQMTQSPSTLSASVGDRVTITCSASSRVGYMHWYQQKPGKAPKLLIYDTSKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCFQGSGYPFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* AP2267 (SEQ ID NO: 8) QVTLRESGPALVKPTQTLTLTCTFSGFSLSTAGMSVGWIRQPPGKALEWLADIWWDDKKHYNPSLKDRLTISKDTSKNQVVLKVTNMDPADTATYYCARDMIFNFYFDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLGGMGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWQDSSFLGGVVHLEAGEKVVVRVLDERLVRLLDGTRSYFGAFMV* AP2269 (SEQ ID NO: 9) QVTLRESGPALVKPTQTLTLTCTFSGFSLSTAGMSVGWIRQPPGKALEWLADIWWDDKKHYNPSLKDRLTISKDTSKNQVVLKVTNMDPADTATYYCARDMIFNFYFDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLGGMGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWQDSSFLGGVVHLEAGEKVVVRVLDERLVRLLDGTRSYFGAFMVGGGGSGGGGSEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLGGMGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWQDSSFLGGVVHLEAGEKVVVRVLDERLVRLLDGTRSYFGAFMV* AP1981 (SEQ ID NO: 10) EVQLQESGPGLVKPSETLSLTCTVSGFSLSIYGVNWVRQPPGKGLEWIAAIWSGGRKDYSLSLKSRLTISGDTSKNQVSLKLSSVTAADTAVYYCARSQDMPGYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLGGVGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEKVVVRVLDERLVRLRDGTRSYFGAFMV AP2464 (SEQ ID NO: 11) E VQLQESGPGLVKPSETLSLTCTVSGFSLSIYGVNWVRQPPGKGLEWIAAIWSGGRKDYSLSLKSRLTISGDTSKNQVSLKLSSVTAADTAVYYCARSQDMPGYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLGGVGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEKVVVRVLDERLVRLRDGTRSYFGAFMVGGGGSGGGGSEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLGGVGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEKVVVRVLDERLVRLRDGTRSYFGAFMV AP2584 (SEQ ID NO: 12) RRSHEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKTGYYYIYSKVQLGGVGCPLGLAGTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEKVVVRVLGKRLVRLRDGTRSYFGAFMV GRRSHEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKTGYYYIYSKVQLGGVGCPLGLAGTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEKVVVRVLGKRLVRLRDGTRSYFGAFMVGRRSHEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKTGYYYIYSKVQLGGVGCPLGLAGTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEKVVVRVLGKRLVRLRDGTRSYFGAFMVGGGGSGGGGSGGGGSGGGGTGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK* AP2586 (SEQ ID NO: 13) EVQLQESGPGLVKPSETLSLTCTVSGFSLSIYGVNWVRQPPGKGLEWIAAIWSGGRKDYSLSLKSRLTISGDTSKNQVSLKLSSVTAADTAVYYCARSQDMPGYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK* AP2585 (SEQ ID NO: 14) RRSHEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKTGYYYIYSKVQLGGVGCPLGLAGTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEKVVVRVLGKRLVRLRDGTRSYFGAFMVGRRSHEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKTGYYYIYSKVQLGGVGCPLGLAGTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEKVVVRVLGKRLVRLRDGTRSYFGAFMVGRRSHEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKTGYYYIYSKVQLGGVGCPLGLAGTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEKVVVRVLGKRLVRLRDGTRSYFGAFMVGGGGSGGGGSGGGGSGGGGTCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK* AP2555 (SEQ ID NO: 15) EVQLQESGPGLVKPSETLSLTCTVSGFSLSIYGVNWVRQPPGKGLEWIAAIWSGGRKDYSLSLKSRLTISGDTSKNQVSLKLSSVTAADTAVYYCARSQDMPGYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSLSPGLPAAHLIGAPLKGQGLGWETTKEQAFLTSGTQFSDAEGLALPQDGLYYLYCLVGYRGRAPPGGGDPQGRSVTLRSSLYRAGGAYGPGTPELLLEGAETVTPVLDPARRQGYGPLWYTSVGFGGLVQLRRGERVYVNISHPDMVDFARGKTFFGAVMVG* AP2556 (SEQ ID NO: 16) EVQLQESGPGLVKPSETLSLTCTVSGFSLSIYGVNWVRQPPGKGLEWIAAIWSGGRKDYSLSLKSRLTISGDTSKNQVSLKLSSVTAADTAVYYCARSQDMPGYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSAHSTLKPAAHLIGDPSKQNSLLWRANTDRAFLQDGFSLSNNSLLVPTSGIYFVYSQVVFSGKAYSPKATSSPLYLAHEVQLFSSQYPFHVPLLSSQKMVYPGLQEPWLHSMYHGAAFQLTQGDQLSTHTDGIPHLVLSPSTVFFGAFALLSPGLPAAHLIGAPLKGQGLGWETTKEQAFLTSGTQFSDAEGLALPQDGLYYLYCLVGYRGRAPPGGGDPQGRSVTLRSSLYRAGGAYGPGTPELLLEGAETVTPVLDPARRQGYGPLWYTSVGFGGLVQLRRGERVYVNISHPDMVDFARGKTFFGAVMVG* The tested immunocytokines are shown in Tables 4 , 4.1 , and 4.2 below. Table 4 : Tested antibodies and heavy/ light chains Protein Concept ID Chain Chain code Protein Concept ID Chain Chain code 9E3 chimera (ABC1139) HC AP1333 ABC1947 LC AP2463 HC1 AP1981 LC AP1334 HC2 AP2464 ABC1931 LC AP2463 ABC2066 LC AP2463 HC1 AP2586 HC AP1992 HC2 AP2584 ABC1930 LC AP2463 ABC2067 LC AP2463 HC1 AP1979 HC1 AP2585 HC2 AP2271 HC2 AP2586 ABC1773 LC AP672 ABC2097 LC AP2463 HC1 AP2267 HC1 AP2555 HC2 AP2269 HC2 AP2556 Table 4.1 : Tested Antibodies and Kabat CDR Protein Concept ID Chain CDR1 CDR2 CDR3 9E3 chimera (ABC1139) HC IYGVN (SEQ ID NO: 26) AIWSGGRKDYNLSLKS (SEQ ID NO: 61) SQDMPGYFDY (SEQ ID NO: 28) LC KTNQNVDYNGNTFMH (SEQ ID NO: 76) LASNLAS (SEQ ID NO: 24) QQSRNLPYT (SEQ ID NO: 25) ABC1931 LC KTNQNVDYSGNTFMH (SEQ ID NO: 79) LASNLAS (SEQ ID NO: 24) QQSRNLPYT (SEQ ID NO: 25) HC IYGVN (SEQ ID NO: 26) AIWSGGRKDYSLSLKS (SEQ ID NO: 63) SQDMPGYFDY (SEQ ID NO: 28) ABC1930 LC KTNQNVDYSGNTFMH (SEQ ID NO: 79) LASNLAS (SEQ ID NO: 24) QQSRNLPYT (SEQ ID NO: 25) HC1 IYGVN (SEQ ID NO: 26) AIWSGGRKDYSLSLKS (SEQ ID NO: 63) SQDMPGYFDY (SEQ ID NO: 28) HC2 IYGVN (SEQ ID NO: 26) AIWSGGRKDYSLSLKS (SEQ ID NO: 63) SQDMPGYFDY (SEQ ID NO: 28) ABC1773 LC SASSRVGYMH (SEQ ID NO: 29) DTSKLAS (SEQ ID NO: 30) FQGSGYPFT (SEQ ID NO: 31) HC1 TAGMSVG (SEQ ID NO: 32) DIWWDDKKHYNPSLKD (SEQ ID NO: 33) DMIFNFYFDV (SEQ ID NO: 34) HC2 TAGMSVG (SEQ ID NO: 32) DIWWDDKKHYNPSLKD (SEQ ID NO: 33) DMIFNFYFDV (SEQ ID NO: 34) ABC1947 LC KTNQNVDYSGNTFMH (SEQ ID NO: 79) LASNLAS (SEQ ID NO: 24) QQSRNLPYT (SEQ ID NO: 25) HC1 IYGVN (SEQ ID NO: 26) AIWSGGRKDYSLSLKS (SEQ ID NO: 63) SQDMPGYFDY (SEQ ID NO: 28) HC2 IYGVN (SEQ ID NO: 26) AIWSGGRKDYSLSLKS (SEQ ID NO: 63) SQDMPGYFDY (SEQ ID NO: 28) ABC2066 LC KTNQNVDYSGNTFMH (SEQ ID NO: 79) LASNLAS (SEQ ID NO: 24) QQSRNLPYT (SEQ ID NO: 25) HC1 / / / HC2 IYGVN (SEQ ID NO: 26) AIWSGGRKDYSLSLKS (SEQ ID NO: 63) SQDMPGYFDY (SEQ ID NO: 28) ABC2067 LC KTNQNVDYSGNTFMH (SEQ ID NO: 79) LASNLAS (SEQ ID NO: 24) QQSRNLPYT (SEQ ID NO: 25) HC1 / / / HC2 IYGVN (SEQ ID NO: 26) AIWSGGRKDYSLSLKS (SEQ ID NO: 63) SQDMPGYFDY (SEQ ID NO: 28) ABC2097 LC KTNQNVDYSGNTFMH (SEQ ID NO: 79) LASNLAS (SEQ ID NO: 24) QQSRNLPYT (SEQ ID NO: 25) HC1 IYGVN (SEQ ID NO: 26) AIWSGGRKDYSLSLKS (SEQ ID NO: 63) SQDMPGYFDY (SEQ ID NO: 28) HC2 IYGVN (SEQ ID NO: 26) AIWSGGRKDYSLSLKS (SEQ ID NO: 63) SQDMPGYFDY (SEQ ID NO: 28) Table 4.2 : Detected antibody and amino acid sequences Chain code Single-code amino acid sequence (variable regions are shown in bold). AP1979 (SEQ ID NO: 5) EVQLQESGPGLVKPSETLSLTCTVSGFSLSIYGVNWVRQPPGKGLEWIAAIWSGGRKDYSLSLKSRLTISGDTSKNQVSLKLSSVTAADTAVYYCARSQDMPGYFDYWGQGTLVTVSS ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQV SLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSEVNPAAHLTGANSSLTGSGGPLLWETQL GLAFLRGLSYHDGALVVTKAGYYYIYSKVQLGGMGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWQDSSFLGGVVHLEAGEKVVVRVLDERLVRLLDGTRSYFGAFMV AP2271 (SEQ ID NO: 6) EVQLQESGPGLVKPSETLSLTCTVSGFSLSIYGVNWVRQPPGKGLEWIAAIWSGGRKDYSLSLKSRLTISGDTSKNQVSLKLSSVTAADTAVYYCARSQDMPGYFDYWGQGTLVTVSS AP672 (SEQ ID NO: 7) DIQMTQSPSTLSASSVGDRVTITCSASSRVGYMHWYQQKPGKAPKLLIYDTSKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCFQGSGYPFTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* AP2267 (SEQ ID NO: 8) QVTLRESGPALVKPTQTLTLTCTFSGFSLSTAGMSVGWIRQPPGKALEWLADIWWDDKKHYNPSLKDRLTISKDTSKNQVVLKVTNMDPADTATYYCARDMIFNFYFDVWGQGTTVTVSS ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVS LSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSEVNPAAHLTGANSSLTGSGGPLLWETQLG LAFLRGLSYHDGALVVTKAGYYYIYSKVQLGGMGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWQDSSFLGGVVHLEAGEKVVVRVLDERLVRLLDGTRSYFGAFMV* AP2269 (SEQ ID NO: 9) QVTLRESGPALVKPTQTLTLTCTFSGFSLSTAGMSVGWIRQPPGKALEWLADIWWDDKKHYNPSLKDRLTISKDTSKNQVVLKVTNMDPADTATYYCARDMIFNFYFDVWGQGTTVTVSS * AP1981 (SEQ ID NO: 10) EVQLQESGPGLVKPSETLSLTCTVSGFSLSIYGVNWVRQPPGKGLEWIAAIWSGGRKDYSLSLKSRLTISGDTSKNQVSLKLSSVTAADTAVYYCARSQDMPGYFDYWGQGTLVTVSS ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQV SLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSEVNPAAHLTGANSSLTGSGGPLLWETQL GLAFLRGLSYHDGALVVTKAGYYYIYSKVQLGGVGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEKVVVRVLDERLVRLRDGTRSYFGAFMV AP2464 (SEQ ID NO: 11) E VQLQESGPGLVKPSETLSLTCTVSGFSLSIYGVNWVRQPPGKGLEWIAAIWSGGRKDYSLSLKSRLTISGDTSKNQVSLKLSSVTAADTAVYYCARSQDMPGYFDYWGQGTLVTVSS AP2584 (SEQ ID NO: 12) RRSHEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKTGYYYIYSKVQLGGVGCPLGLAGTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEKVVVRVLGKRLVRLRDGTRSYFGAFMV G * AP2586 (SEQ ID NO: 13) EVQLQESGPGLVKPSETLSLTCTVSGFSLSIYGVNWVRQPPGKGLEWIAAIWSGGRKDYSLSLKSRLTISGDTSKNQVSLKLSSVTAADTAVYYCARSQDMPGYFDYWGQGTLVTVSS ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK* AP2585 (SEQ ID NO: 14) * AP2555 (SEQ ID NO: 15) EVQLQESGPGLVKPSETLSLTCTVSGFSLSIYGVNWVRQPPGKGLEWIAAIWSGGRKDYSLSLKSRLTISGDTSKNQVSLKLSSVTAADTAVYYCARSQDMPGYFDYWGQGTLVTVSS ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSLSPGLPAAHLIGAPLKGQGLGWETTKEQAFLTSGTQFS DAEGLALPQDGLYYLYCLVGYRGRAPPGGGDPQGRSVTLRSSLYRAGGAYGPGTPELLLEGAETVTPVLDPARRQGYGPLWYTSVGFGGLVQLRRGERVYVNISHPDMVDFARGKTFFGAVMVG* AP2556 (SEQ ID NO: 16) EVQLQESGPGLVKPSETLSLTCTVSGFSLSIYGVNWVRQPPGKGLEWIAAIWSGGRKDYSLSLKSRLTISGDTSKNQVSLKLSSVTAADTAVYYCARSQDMPGYFDYWGQGTLVTVSS *

圖2及圖6顯示上述免疫細胞介素的形式。形式A：ABC2066、ABC2067；形式D：ABC1930、ABC1773、ABC1947、及ABC2097。Figures 2 and 6 show the forms of the above-mentioned immune intercellular molecules. Form A: ABC2066, ABC2067; Form D: ABC1930, ABC1773, ABC1947, and ABC2097.

在該研究中，將三個串聯連接的人類突變體LIGHT (human mutant LIGHT, hmLIGHT)單元連接到Fc區之N端以與抗FAP人源化9E3半體組裝。結構（形式A）及hmLIGHT突變係基於先前報導的免疫細胞介素(Cancer Cell 29, 285-296, 2016)。然而，在hIgG4 Fc起始殘基中略有差異的兩種形式A的構築體（ABC2066及ABC2067）未能正確組裝，這是因為編碼三個串聯hmLIGHT單元的鏈表現出低表現，即使具有不同的比率。In this study, three tandemly linked human mutant LIGHT (hmLIGHT) units were attached to the N-terminus of the Fc region to assemble with the anti-FAP humanized 9E3 hemisome. The structures (form A) and the hmLIGHT mutant were based on previously reported immunocytokines (Cancer Cell 29, 285-296, 2016). However, the two form A constructs (ABC2066 and ABC2067) with slight differences in the hIgG4 Fc initiation residue failed to assemble correctly because the chains encoding the three tandem hmLIGHT units exhibited low performance, even with different ratios.

相比之下，與ABC2066及ABC2067共享相同抗FAP可變域的呈形式D之ABC1930及ABC1947展示正確的組裝。基於LIGHT的免疫細胞介素的成功表現及正確組裝藉由非還原性凝膠中存在與正確組裝分子的分子量相對應的顯性條帶來證明。由還原蛋白的SDS-PAGE分析揭示的三個條帶進一步支持了正確的組裝，該三個條帶代表兩條不同分子量的重鏈及一條輕鏈（圖6）。In contrast, ABC1930 and ABC1947, which share the same anti-FAP variable domain as ABC2066 and ABC2067, exhibited correct assembly in form D. The successful performance and correct assembly of LIGHT-based immunocytokines were demonstrated by the presence of dominant bands in the non-reducing gel corresponding to the molecular weight of the correctly assembled molecules. Three bands revealed by SDS-PAGE analysis of the reduced proteins further supported correct assembly, representing two heavy chains of different molecular weights and one light chain (Figure 6).

此外，除了所欲的受體選擇性外，具有LIGHT1突變體（縮寫為mu1或LIGHTmu1或LIGHT1）的ABC1930展現出比具有野生型LIGHT（縮寫為wt或LIGHTwt）的ABC1947高3倍的表現（圖7A及圖7B）。In addition to the desired receptor selectivity, ABC1930 with the LIGHT1 mutant (abbreviated as mu1 or LIGHTmu1 or LIGHT1) exhibited 3 times higher performance than ABC1947 with wild-type LIGHT (abbreviated as wt or LIGHTwt) (Figs. 7A and 7B).

與LIGHTmu1相比，淋巴毒素-αββ對LTßR具有與HVEM及DcR3相似的受體結合選擇性，在提供抗腫瘤效果方面潛在地提供增強的安全性及功效。ABC2097係一種具有呈形式D的淋巴毒素-αββ的免疫細胞介素，亦可產生表現，儘管產率較低（圖7A及圖7B）。Compared to LIGHTmu1, lymphotoxin-αββ exhibits receptor-binding selectivity for LTβR similar to that of HVEM and DcR3, potentially offering enhanced safety and efficacy in providing antitumor effects. ABC2097, an immunocytokine possessing lymphotoxin-αββ in form D, also produces efficacies, albeit at a lower yield (Figures 7A and 7B).

ABC1930中的LIGHTmu1突變體具有如SEQ ID NO: 17所示之胺基酸序列，且ABC1947中的野生型LIGHT具有如SEQ ID NO: 18所示之胺基酸序列。 2.2.由BLI測定的FAP結合 The LIGHTmu1 mutant in ABC1930 has the amino acid sequence shown in SEQ ID NO: 17, and the wild-type LIGHT in ABC1947 has the amino acid sequence shown in SEQ ID NO: 18. 2.2. FAP binding determined by BLI

在30℃下在Octet RH16系統(Sartorius)上執行動力學檢定，且在PBST緩衝液（含0.02% Tween-20的PBS，pH 7.4）中以1000 rpm的軌道振動速度稀釋樣本。為了評估ABC1930與FAP重組蛋白質之間的結合動力學，將Ni-NTA生物感測器(Sartorius, 18-5101)加載10 ug/mL His標記的FAP抗原蛋白質（人類或食蟹獼猴或小鼠）至1.0 nm的密度，隨後在PBST緩衝液中進行60秒的基線步驟。接著將FAP捕獲的生物感測器浸入在含有不同濃度ABC1930的孔中2分鐘，隨後在PBST緩衝液中解離4分鐘。亦將FAP捕獲的感測器浸入含有PBST緩衝液的孔中，以允許單一參考扣除，以補償捕獲的FAP的自然解離。感測圖以緩衝液為空白參考，且用1:1模型進行全域擬合，以使用Data Analysis軟體(Fortebio/Sartorius)提取動力學參數。參見圖3A及表3。 2.3由BLI測定的LTßR及HVEM結合 Kinetic assays were performed at 30°C on an Octet RH16 system (Sartorius), with samples diluted in PBST buffer (PBS containing 0.02% Tween-20, pH 7.4) at a track vibration speed of 1000 rpm. To evaluate the binding dynamics between ABC1930 and FAP recombinant proteins, a Ni-NTA biosensor (Sartorius, 18-5101) was loaded with 10 ug/mL His-labeled FAP antigen protein (human, cynomolgus monkey, or mouse) to a density of 1.0 nm, followed by a baseline step in PBST buffer for 60 seconds. Next, the FAP-captured biosensors were immersed in wells containing different concentrations of ABC1930 for 2 minutes, followed by dissociation in PBST buffer for 4 minutes. The FAP-captured biosensors were also immersed in wells containing PBST buffer to allow for single-reference subtraction to compensate for the natural dissociation of captured FAP. Sensing plots were performed globally using the buffer as a blank reference and a 1:1 model was used to extract kinetic parameters using Data Analysis software (Fortebio/Sartorius). See Figure 3A and Table 3. 2.3 LTßR and HVEM Combination Measured by BLI

使用EZ-Link Sulfo-NHS-生物素套組(#21925, Thermo scientific)對LTßR-Fc蛋白質進行生物素化。將Streptavidin Biosensor (ForteBio)浸泡在PBST緩衝液中進行檢定。首先，將感測器在PBST緩衝液中沖洗180秒，作為基線。其次，用經生物素化目標蛋白(20 µg/ml)將感測器固定200秒。接著，將感測器在PBST緩衝液中再洗滌60秒。最後，將感測器暴露於一系列稀釋的免疫細胞介素，最大濃度為100 nM。監測各濃度的免疫細胞介素的締合180秒，隨後在PBST緩衝液中解離180秒。為了洗去免疫細胞介素，將SA生物感測器浸入3 M MgCl 2中10秒，重複3次。感測圖以緩衝液為空白參考，且用1:1模型進行全域擬合，以使用Data Analysis軟體(Fortebio/Sartorius)提取動力學參數。 The LTßR-Fc protein was biotinylated using the EZ-Link Sulfo-NHS-Biotin kit (#21925, Thermo Scientific). The Streptavidin Biosensor (ForteBio) was then immersed in PBST buffer for assay. First, the sensor was rinsed in PBST buffer for 180 seconds as a baseline. Next, the sensor was fixed with the biotinylated target protein (20 µg/ml) for 200 seconds. Then, the sensor was rinsed again in PBST buffer for 60 seconds. Finally, the sensor was exposed to a series of diluted immunocytokines, up to a maximum concentration of 100 nM. The binding of immunocytokines at various concentrations was monitored for 180 seconds, followed by dissociation in PBST buffer for 180 seconds. To wash away the immunocytokines, the SA biosensor was immersed in 3 M MgCl2 for 10 seconds, repeated 3 times. The sensor map was used as a blank reference with the buffer and fitted globally using a 1:1 model to extract kinetic parameters using Data Analysis software (Fortebio/Sartorius).

HVEM-His蛋白質係購自Sino Biological（目錄編號10334-H08H、90109-C08B、10567-M08H）。將Ni-NTA生物感測器(Sartorius, 18-5101)加載10 ug/mL His標記的HVEM蛋白質（人類或食蟹獼猴或小鼠）至1.0 nm的密度，隨後在PBST緩衝液中進行60秒基線步驟。接著將生物感測器浸入含有不同濃度ABC1930或ABC1947的孔中2分鐘，隨後在PBST緩衝液中解離4分鐘。為了解釋捕獲的HVEM的解離，亦將感測器浸入含有PBST緩衝液的孔中以進行單一參考扣除。感測圖以緩衝液為空白參考，且用1:1模型進行全域擬合，以使用Data Analysis軟體(Fortebio/Sartorius)提取動力學參數。參見圖3B至圖3C及表3。 2.4由ELISA測定的DcR3結合 The HVEM-His protein was purchased from Sino Biological (catalog numbers 10334-H08H, 90109-C08B, 10567-M08H). A Ni-NTA biosensor (Sartorius, 18-5101) was loaded with 10 μg/mL His-labeled HVEM protein (human, cynomolgus monkey, or mouse) to a density of 1.0 nm, followed by a 60-second baseline step in PBST buffer. The biosensor was then immersed in wells containing different concentrations of ABC1930 or ABC1947 for 2 minutes, followed by dissociation in PBST buffer for 4 minutes. To explain the dissociation of captured HVEMs, the sensor was also immersed in a well containing PBST buffer for single-reference subtraction. Sensing plots were performed globally using the buffer as a blank reference and a 1:1 model was used to extract kinetic parameters using Data Analysis software (Fortebio/Sartorius). See Figures 3B to 3C and Table 3. 2.4 DcR3 binding as determined by ELISA

DcR3係在人類及食蟹獼猴中表現但在小鼠中不存在的可溶性蛋白質。DcR3負向調控LIGHT活化LTβR及HVEM的能力(Liu et al., 2021)，且在某些自體發炎性疾病及癌症患者中表現上調(Fuchsberger et al., 2021)。與DcR3與LIGHTmu1之結合親和力低於野生型LIGHT一致，DcR3與ABC1930的結合顯著弱於與ABC1947之結合。DcR3 is a soluble protein present in humans and cynomolgus monkeys but absent in mice. DcR3 negatively regulates the ability of LIGHT to activate LTβR and HVEM (Liu et al., 2021), and is upregulated in some autoinflammatory diseases and cancer patients (Fuchsberger et al., 2021). Consistent with the lower binding affinity of DcR3 to LIGHTmu1 compared to wild-type LIGHT, the binding of DcR3 to ABC1930 is significantly weaker than its binding to ABC1947.

藉由將人類DcR3殘基33-300融合至兔Fc之N端來產生重組DcR3蛋白。重組蛋白質在Expi293F中表現且由蛋白質A柱進行親和純化，且基於Superdex 200收集單體。Recombinant DcR3 protein was generated by fusing human DcR3 residue 33-300 to the N-terminus of rabbit Fc. The recombinant protein was expressed in Expi293F and affinity purified by protein A-pillar, and monomers were collected using Superdex 200.

將DcR3蛋白以2 µg/mL的濃度固定在maxiSorp 96孔ELISA盤(Thermo Fisher)上，且接著用0.5% BSA-PBST緩衝液阻斷一小時。將生物素化的基於LIGHT的免疫細胞介素（EZ-Link Sulfo-NHS-生物素套組，#21925，Thermo Scientific, Rockford, IL, USA）以各種稀釋度添加到盤中，最大濃度為10 nM。將盤培養一小時，用PBST洗滌四次，且進一步與HRP接合的鏈黴抗生物素(Acrobiosystems, #STN-NH913)一起培養，且再次用PBST洗滌。將TMB受質(#34029，Thermo Fisher)添加到盤中，且在SpectraMax M5微量盤讀取儀(Molecular Devices)上以450 nM讀取盤。參見圖4。DcR3 protein was immobilized at a concentration of 2 µg/mL on a maxiSorp 96-well ELISA plate (Thermo Fisher) and then buffered with 0.5% BSA-PBST for one hour. Biotinylated LIGHT-based immunocytokines (EZ-Link Sulfo-NHS-Biotin Kit, #21925, Thermo Scientific, Rockford, IL, USA) were added to the plates at various dilutions, up to a maximum concentration of 10 nM. The plates were incubated for one hour, washed four times with PBST, and then further incubated with HRP-conjugated strepto-acid (Acrobiosystems, #STN-NH913) and washed again with PBST. Add the TMB substrate (#34029, Thermo Fisher) to the disk and read the disk at 450 nM using a SpectraMax M5 Molecular Devices reader. See Figure 4.

FAP、LTßR、HVEM、及DcR3結合的結果係彙總於表3。 表3 ：FAP 、LTßR 、HVEM 、及DcR3 結合 ABC1930 ABC1947 目標 FAP LTßR HVEM DcR3(ELISA) LTßR HVEM DcR3(ELISA) 物種 K D(nM) K D(nM) K D(nM) EC50 (nM) K D(nM) K D(nM) EC50 (nM) 人類 3.0 1.2 無結合 減少 0.6 1.3 0.056 食蟹獼猴 4.7 1.8 無結合 減少 1.1 1.0 0.044 小鼠 2.6 4.3 142 nM NA 8 4.8 NA The results of combining FAP, LTßR, HVEM, and DcR3 are summarized in Table 3. Table 3 : Combinations of FAP , LTßR , HVEM , and DcR3 ABC1930 ABC1947 Target FAP LTßR HVEM DcR3(ELISA) LTßR HVEM DcR3(ELISA) species K D (nM) K D (nM) K D (nM) EC50 (nM) K D (nM) K D (nM) EC50 (nM) human 3.0 1.2 Unbound Reduce 0.6 1.3 0.056 Crab-eating macaques 4.7 1.8 Unbound Reduce 1.1 1.0 0.044 mice 2.6 4.3 142 nM NA 8 4.8 NA

ABC1930與LTßR結合而不與HVEM及DcR3接合，潛在地在提供抗腫瘤效果方面提供增強的安全性及功效。 2.5由ELISA測定的DPPIV結合 ABC1930 binds to LTßR but not to HVEM and DcR3, potentially offering enhanced safety and efficacy in providing antitumor effects. 2.5 DPPIV binding as determined by ELISA

與人類FAP相似性最高的蛋白質係DPPIV。為了測試ABC1930之特異性，執行DPPIV ELISA。The protein most similar to human FAP is DPPIV. To test the specificity of ABC1930, a DPPIV ELISA was performed.

人類FAP及DPPIV蛋白係以0.5 µg/mL的濃度固定在MaxiSorp 96孔ELISA盤上，接著用0.5% BSA-PBST緩衝液阻斷一小時。將ABC1930以一系列稀釋度添加到盤中，最大濃度為50 nM。將盤培養一小時，用PBST洗滌四次，且進一步與HRP接合的驢抗人類IgG (H+L)抗體(Jackson Immunoresearch, #709-035-149)一起培養，隨後用PBST再洗滌四次。將TMB受質(#34029，Thermo Fisher)添加到盤中，且在SpectraMax M5微量盤讀取儀(Molecular Devices)上以450 nM讀取盤。Human FAP and DPPIV proteins were immobilized at a concentration of 0.5 µg/mL on MaxiSorp 96-well ELISA plates, followed by one hour of inhibition with 0.5% BSA-PBST buffer. ABC1930 was added to the plates at a series of dilutions, with a maximum concentration of 50 nM. The plates were incubated for one hour, washed four times with PBST, and further cultured with HRP-conjugated donkey anti-human IgG (H+L) antibody (Jackson Immunoresearch, #709-035-149), followed by four more washes with PBST. Add the TMB substrate (#34029, Thermo Fisher) to the disk and read the disk at 450 nM on a SpectraMax M5 Molecular Devices micro disk reader.

如圖5所顯示，ABC1930與人類FAP結合，但不與DPPIV結合。 實例3 ：使用經工程改造細胞系的功能檢定3.1評估ABC1931、ABC1773、及ABC1930對內源小鼠LTβR路徑之順式活化，其使用CCL2作為替代讀出 As shown in Figure 5, ABC1930 binds to human FAP but not to DPPIV. Example 3 : Functional assay using engineered cell lines 3.1 Evaluation of cis-activation of the endogenous mouse LTβR pathway by ABC1931, ABC1773, and ABC1930 using CCL2 as a surrogate readout

使用兩種小鼠細胞系BALB/c-3T3-FAP (LTβR+ HVEM- FAP+)及BALB/c-3T3 (LTβR+ HVEM- FAP-)研究ABC1931、ABC1773、及ABC1930對活化內源小鼠LTβR之效應。藉由用小鼠FAP（縮寫為mFAP）轉染BALB/c-3T3細胞(ATCC)產生BALB/c-3T3-FAP細胞系，選擇且確認該細胞系的mFAP表現。將細胞以每孔4,000個細胞接種於96孔組織培養盤中，且與不同濃度的抗FAP-LIGHT免疫細胞介素在37℃下培養24小時。離心之後，收集上清液以由具有Tecan Spark讀取儀的ELISA（目錄號432704，Biolegend）定量CCL2蛋白之水平。The effects of ABC1931, ABC1773, and ABC1930 on activated endogenous mouse LTβR were investigated using two mouse cell lines, BALB/c-3T3-FAP (LTβR+ HVEM- FAP+) and BALB/c-3T3 (LTβR+ HVEM- FAP-). BALB/c-3T3-FAP cell lines were generated by transfecting BALB/c-3T3 cells (ATCC) with mouse FAP (mFAP), and mFAP expression in these cell lines was selected and confirmed. Cells were seeded at 4,000 cells per well in 96-well tissue culture dishes and cultured at 37°C for 24 hours with different concentrations of anti-FAP-LIGHT immunocytokines. After centrifugation, the supernatant was collected to quantify the level of CCL2 protein using an ELISA (catalog number 432704, Biolegend) with a Tecan Spark reader.

圖8A至圖8C顯示無論FAP表現如何，ABC1931均不誘導CCL2產生。然而，當細胞表現FAP及LTβR兩者時，ABC1930更顯著地活化LTβR信號傳導(＞1000x)，表明存在順式活化。非標靶對照ABC1773（抗FAP-LIGHTmu1）未能展現該順式活化。由於腫瘤微環境中的癌相關纖維母細胞(cancer-associate fibroblast, CAF)在腫瘤生物學中發揮重要作用，且具有共表現FAP及LTβR兩者的潛力，因此ABC1930將優先刺激腫瘤微環境中FAP+ LTβR + CAF中的LTβR。 3.2評估ABC1931、ABC1773、及ABC1930對內源小鼠LTβR路徑之反式活化，其使用CCL2作為替代讀出 Figures 8A to 8C show that ABC1931 does not induce CCL2 production regardless of FAP expression. However, when cells express both FAP and LTβR, ABC1930 significantly activates LTβR signaling (>1000x), indicating cis activation. The non-target control ABC1773 (anti-FAP-LIGHTmu1) failed to exhibit this cis activation. Since cancer-associated fibroblasts (CAFs) in the tumor microenvironment play a crucial role in tumor biology and have the potential to co-express both FAP and LTβR, ABC1930 will preferentially stimulate LTβR in FAP + LTβR + CAF within the tumor microenvironment. 3.2 The transactivation of the endogenous mouse LTβR pathway by ABC1931, ABC1773, and ABC1930 was evaluated, using CCL2 as a surrogate readout.

為了研究反式表現的FAP對BALB/c-3T3-WT (LTβR+ HVEM- FAP-)細胞中內源LTβR路徑活化之效應，藉由使用lipofectamine 3000及抗生素選擇用含有小鼠FAP基因的質體穩定轉染CT26細胞(ATCC)來產生CT26-FAP細胞系。本發明人證實，在基於LIGHT的免疫細胞介素刺激之後，僅3T3細胞而非CT26-WT或CT26-FAP可表現CCL2。To investigate the effect of trans-expressed FAP on the activation of the endogenous LTβR pathway in BALB/c-3T3-WT (LTβR+ HVEM- FAP-) cells, the CT26-FAP cell line was generated by selectively transfecting CT26 cells (ATCC) with plasmids containing the mouse FAP gene using lipofectamine 3000 and antibiotics. The inventors demonstrated that, following LIGHT-based immune cytokine stimulation, only 3T3 cells, not CT26-WT or CT26-FAP, expressed CCL2.

將CT26-WT或CT26-FAP細胞以10,000/孔接種在96孔組織培養盤中，且與連續稀釋的ABC1931、ABC1773、及ABC1930在37℃、5% CO 2下一起培養一小時。接著將4,000個BALB/c-3T3細胞作為應答細胞添加到培養物中。執行ELISA檢定（目錄號432704，Biolegend），以測量培養基中之CCL2蛋白水平作為LTβR路徑活化之替代讀出。 CT26-WT or CT26-FAP cells were seeded at 10,000 cells/well in 96-well tissue culture dishes and co-cultured with serially diluted ABC1931, ABC1773, and ABC1930 at 37°C and 5% CO2 for one hour. Then, 4,000 BALB/c-3T3 cells were added to the culture as responders. An ELISA assay (catalog number 432704, Biolegend) was performed to measure CCL2 protein levels in the medium as a surrogate read for LTβR pathway activation.

如圖9A至圖9C所顯示，在CT26-FAP細胞存在下，ABC1930更顯著地活化LTβR信號傳導(＞100x)，表明存在反式活化。非標靶對照ABC1773（對照抗體-LIGHTmu1）未能展現該反式活化。 實例4 ：KPC 小鼠模型中之體內評估 As shown in Figures 9A to 9C, in the presence of CT26-FAP cells, ABC1930 significantly activated LTβR signaling (>100x), indicating transactivation. The non-target control ABC1773 (control antibody-LIGHTmu1) failed to exhibit this transactivation. Example 4 : In vivo evaluation in a KPC mouse model.

KPC0826細胞系來源於市售的KPC細胞系，該KPC細胞系係衍生自基因改造的KPC腫瘤模型。KPC0826係衍生自該商業細胞系使用先前發布的規程建立的侵襲性KPC腫瘤塊(Beatty et al., 2011)。為了建立皮下KPC0826腫瘤模型，將50 µl HBSS中的250萬個KPC0826細胞與50 µl基質膠混合並植入C57BL/6小鼠的側腹中。建立的皮下KPC0826腫瘤模型顯示FAP+ CAF細胞相對較高的存在，約占腫瘤組織總解離細胞的1至5%（數據未顯示）。在第0天，當腫瘤體積達到平均約113 mm 3時，基於體重及腫瘤體積將小鼠分為6組：G1_緩衝液、G2_GEM（吉西他濱）、G3_ABC1931、G4_ABC1931 + GEM、G5_ABC1930、及G6_ABC1930 + GEM，每組六隻小鼠。用0.75奈莫耳的各別大分子每週治療小鼠兩次，共7個劑量，對應於ABC1931的5.4 mg/kg及ABC1930的7.25 mg/kg的劑量。GEM係以75 mg/kg給予。藉由在指定時間點使用卡尺測量腫瘤體積來監測腫瘤生長，直到第24天。使用下式計算腫瘤體積：長x寬 2/2。將腫瘤生長抑制(tumor growth inhibition, TGI)與對照組（G1_緩衝液）比較，且使用DRAP R軟體包進行計算(J Transl Med 17, 39, 2019)。 The KPC0826 cell line was derived from a commercially available KPC cell line derived from a genetically modified KPC tumor model. The KPC0826 line was derived from an invasive KPC tumor mass established using this commercial cell line according to previously published procedures (Beatty et al., 2011). To establish a subcutaneous KPC0826 tumor model, 2.5 million KPC0826 cells in 50 µl of HBSS were mixed with 50 µl of matrix gel and implanted into the flank of C57BL/6 mice. The established subcutaneous KPC0826 tumor model showed a relatively high presence of FAP+ CAF cells, accounting for approximately 1% to 5% of the total dissociated cells in the tumor tissue (data not shown). On day 0, when the tumor volume reached an average of approximately 113 mm³ , mice were randomly assigned to six groups based on body weight and tumor volume: G1_blended fluid, G2_GEM (gemcitabine), G3_ABC1931, G4_ABC1931 + GEM, G5_ABC1930, and G6_ABC1930 + GEM, with six mice in each group. Mice were treated twice weekly with 0.75 nanomolar of each macromolecule for a total of seven doses, corresponding to 5.4 mg/kg of ABC1931 and 7.25 mg/kg of ABC1930. GEM was administered at 75 mg/kg. Tumor growth was monitored by measuring tumor volume with calipers at specified time points until day 24. Tumor volume was calculated using the formula: length x width² /2. Tumor growth inhibition (TGI) was compared with the control group (G1 buffer), and the calculations were performed using the DRAP R software package (J Transl Med 17, 39, 2019).

如圖10A至圖10C所顯示，結果表明，ABC1930在KPC0826模型中表現出良好的單藥抗腫瘤活性，TGI達到80%以上。ABC1930與GEM的組合進一步抑制了腫瘤生長。 實例5 ：LL2 腫瘤模型中之體內評估 As shown in Figures 10A to 10C, the results indicate that ABC1930 exhibited good single-agent antitumor activity in the KPC0826 model, with a TGI exceeding 80%. The combination of ABC1930 and GEM further inhibited tumor growth. Example 5 : In vivo evaluation in the LL2 tumor model

為了建立皮下LL2腫瘤模型，將50 µl HBSS中的12萬個LL2細胞與50 µl基質膠混合並植入C57LB/6小鼠的側腹中。在第0天，當腫瘤體積達到平均約40 mm 3時，基於體重及腫瘤體積將小鼠分為3組：G1_Ctrl（媒劑）、G2_ABC1930（抗FAP-LIGHT mu1）、G3_ABC1773（對照抗體-LIGHT mu1），每組六隻小鼠。用0.25奈莫耳的各別大分子每週治療小鼠兩次，共3個劑量，對應於ABC1930及ABC1773的2.417 mg/kg的劑量。藉由在指定時間點使用卡尺測量腫瘤體積來監測腫瘤生長，直到第12天。使用下式計算腫瘤體積：長x寬 2/2。 To establish a subcutaneous LL2 tumor model, 120,000 LL2 cells from 50 µl of HBSS were mixed with 50 µl of matrix gel and implanted into the flank of C57LB/6 mice. On day 0, when the tumor volume reached an average of approximately 40 mm³ , the mice were divided into three groups based on body weight and tumor volume: G1_Ctrl (mediator), G2_ABC1930 (anti-FAP-LIGHT mu1), and G3_ABC1773 (control antibody-LIGHT mu1), with six mice in each group. Mice were treated twice weekly with 0.25 nanomolar of each macromolecule for a total of three doses, corresponding to a dose of 2.417 mg/kg for ABC1930 and ABC1773. Tumor growth was monitored by measuring tumor volume with calipers at specified time points until day 12. The tumor volume was calculated using the following formula: length x width² /2.

結果表明，ABC1930免疫細胞介素在LL2模型中表現出顯著的單藥抗腫瘤活性，比非標靶對照分子ABC1773更有效。The results showed that ABC1930 immunocytokines exhibited significant single-drug antitumor activity in the LL2 model, and were more effective than the non-target control molecule ABC1773.

圖11A至圖11C展現皮下LL2模型中ABC1930抗FAP-LIGHT mu1之體內抗腫瘤活性。圖11A概述治療時間軸。圖11B顯示各組的個體腫瘤體積。圖11C展示各組之平均腫瘤體積。 實例6 ：LL2-OVA 腫瘤模型中之體內評估 Figures 11A to 11C illustrate the in vivo antitumor activity of ABC1930 against FAP-LIGHT mu1 in the subcutaneous LL2 model. Figure 11A outlines the treatment timeline. Figure 11B shows the individual tumor volume in each group. Figure 11C shows the mean tumor volume in each group. Example 6 : In vivo assessment in the LL2-OVA tumor model.

為了建立皮下LL2-OVA腫瘤模型，將50 µl HBSS中的12萬個LL2-OVA細胞與50 µl基質膠混合並植入C57LB/6小鼠的側腹中。在第0天，當腫瘤體積達到平均約80 mm 3時，基於體重及腫瘤體積將小鼠分為4組：G1_Ctrl（媒劑）、G2_ABC092（抗PD1）、G3_ABC1930（抗FAP-LIGHT mu1）、G4_ABC1930（抗FAP-LIGHT mu1）+ ABC092（抗PD1），每組五隻小鼠。在第0天及第5天對小鼠進行治療，共2個劑量。藉由在指定時間點使用卡尺測量腫瘤體積來監測腫瘤生長，直到第8天。使用下式計算腫瘤體積：長x寬 2/2。 To establish a subcutaneous LL2-OVA tumor model, 120,000 LL2-OVA cells from 50 µl of HBSS were mixed with 50 µl of matrix gel and implanted into the flank of C57LB/6 mice. On day 0, when the tumor volume reached an average of approximately 80 mm³ , the mice were divided into four groups based on body weight and tumor volume: G1_Ctrl (mediator), G2_ABC092 (anti-PD1), G3_ABC1930 (anti-FAP-LIGHT mu1), and G4_ABC1930 (anti-FAP-LIGHT mu1) + ABC092 (anti-PD1), with five mice in each group. The mice were treated with two doses on days 0 and 5. Tumor growth was monitored by measuring tumor volume with calipers at specified time points until day 8. The tumor volume was calculated using the following formula: length x width² /2.

ABC092係衍生自於公認且廣泛使用的抗小鼠抗PD1抗體（稱為殖株RPM1-14）。ABC092之重鏈包括如SEQ ID NO: 19所示之胺基酸序列，ABC 092之輕鏈包括如SEQ ID NO: 20所示之胺基酸序列。ABC092 is derived from a recognized and widely used anti-mouse anti-PD1 antibody (referred to as strain RPM1-14). The heavy chain of ABC092 includes the amino acid sequence shown in SEQ ID NO: 19, and the light chain of ABC092 includes the amino acid sequence shown in SEQ ID NO: 20.

單獨的ABC092抗PD1在短的治療時段內未顯示出顯著效果。相反，ABC1930表現出顯著的單藥活性及與抗PD1的輕微協同作用。ABC092 alone did not show significant efficacy against PD-1 in a short treatment period. In contrast, ABC1930 showed significant monotherapy activity and mild synergistic effect with anti-PD-1.

圖12A至圖12C展現皮下LL2-OVA模型中ABC1930抗FAP-LIGHT mu1的體內抗腫瘤活性。圖12A概述治療時間軸及劑量。圖12B顯示各組的個體腫瘤體積。圖12C展示各組之平均腫瘤體積。 實例7 ：皮下EMT6 腫瘤模型中之體內評估 Figures 12A to 12C illustrate the in vivo antitumor activity of ABC1930 against FAP-LIGHT mu1 in the subcutaneous LL2-OVA model. Figure 12A summarizes the treatment timeline and dosage. Figure 12B shows the individual tumor volume in each group. Figure 12C shows the mean tumor volume in each group. Example 7 : In vivo assessment in the subcutaneous EMT6 tumor model.

為了建立皮下EMT6腫瘤模型，將50 µl HBSS中的50萬個EMT6細胞與50 µl基質膠混合並植入BALB/c小鼠的側腹中。在第0天，當腫瘤體積達到平均約92 mm 3時，基於體重及腫瘤體積將小鼠分為4組：G1_媒劑（對照）、G2_ABC1930（抗FAP-LIGHT mu1）、G3_ABC727（抗PDL1）、G4_ABC1930（抗FAP-LIGHT mu1）+ ABC727（抗PDL1），每組六隻小鼠。每週治療小鼠兩次，共4個劑量。藉由在指定時間點使用卡尺測量腫瘤體積來監測腫瘤生長，直到第14天。使用下式計算腫瘤體積：長x寬 2/2。 To establish a subcutaneous EMT6 tumor model, 500,000 EMT6 cells from 50 µl of HBSS were mixed with 50 µl of matrix gel and implanted into the flank of BALB/c mice. On day 0, when the tumor volume reached an average of approximately 92 mm³ , mice were divided into four groups based on body weight and tumor volume: G1_mediator (control), G2_ABC1930 (anti-FAP-LIGHT mu1), G3_ABC727 (anti-PDL1), and G4_ABC1930 (anti-FAP-LIGHT mu1) + ABC727 (anti-PDL1), with six mice in each group. Mice were treated twice a week for a total of four doses. Tumor growth was monitored by measuring tumor volume with calipers at specified time points until day 14. Calculate tumor volume using the following formula: length x width² /2.

ABC727係衍生自Roche的阿替利珠單抗，具有包括如SEQ ID NO: 21所示之胺基酸序列的重鏈、及包括如SEQ ID NO: 22所示之胺基酸序列的輕鏈。ABC727 is an atezolizumab derived from Roche, having a heavy chain including the amino acid sequence shown in SEQ ID NO: 21 and a light chain including the amino acid sequence shown in SEQ ID NO: 22.

在EMT6皮下模型中，ABC1930及抗PDL1兩者皆展現出單一藥劑活性。該組合進一步加深了抗腫瘤反應。因此，到第14天，抗PDL1組中6個腫瘤中有1個消退，且在組合組中6個腫瘤中有2個消退。In the EMT6 subcutaneous model, both ABC1930 and anti-PDL1 exhibited single-drug activity. This combination further enhanced the antitumor response. Consequently, by day 14, one of the six tumors in the anti-PDL1 group and two of the six tumors in the combination group had regressed.

圖13A至圖13C展現皮下EMT6模型中ABC1930抗FAP-LIGHT mu1中之體內抗腫瘤活性。圖13A概述治療時間軸及劑量。圖13B顯示各組的個體腫瘤體積。圖13C展示各組之平均腫瘤體積。Figures 13A to 13C illustrate the in vivo antitumor activity of ABC1930 against FAP-LIGHT mu1 in the subcutaneous EMT6 model. Figure 13A summarizes the treatment timeline and dosage. Figure 13B shows the individual tumor volume in each group. Figure 13C shows the mean tumor volume in each group.

無without

以下係對圖式的簡要描述，該等圖式係出於說明本文揭示的例示性實施例的目的而非出於限制該等例示性實施例的目的而呈現的。 ［圖1A］至［圖1C］顯示如藉由FACS偵測之9E3嵌合體及ABC1931之細胞結合曲線。 ［圖2］顯示抗FAP-LIGHT融合蛋白ABC1930之示意性結構。 ［圖3A］至［圖3B］顯示使用ABC1930之連續稀釋液的固定化FAP或LTßR ECD蛋白之生物膜干涉技術(biolayer interferometry, BLI)感測圖。［圖3C］繪示使用ABC1947或ABC1930之連續稀釋液的固定化HVEM ECD蛋白之BLI感測圖。 ［圖4］顯示如藉由ELISA偵測之ABC1930或ABC1947與經塗佈DcR3蛋白之結合。 ［圖5］顯示如藉由ELISA偵測之ABC1930與經塗佈人類FAP或DPPIV蛋白之結合。 ［圖6］顯示實例2.1中產生之融合蛋白之示意性形式A及D。SDS-PAGE結果指示，具有LIGHTmu1或LIGHTwt之形式D經正確組裝，而形式A中編碼三個串聯hmLIGHT單元之鏈展現出低表現，導致兩個構築體組裝不良。 ［圖7A］顯示在蛋白質A純化之後ABC2097、ABC1930、及ABC1947之產率比較。［圖7B］展示此等蛋白質之SDS-PAGE凝膠影像。 ［圖8A］至［圖8C］分別顯示由ABC1931、ABC1773、及ABC1930自3T3細胞系及3T3-FAP細胞系兩者誘導的CCL2產生。 ［圖9A］至［圖9C］分別顯示在用ABC1931、ABC1773、或ABC1930治療後，CT26-FAP細胞對3T3細胞中內源LTβR路徑之反式活化，其使用CCL2作為替代讀出。 ［圖10A］顯示KPC小鼠模型中用於體內評估的投予排程。［圖10B］至［圖10C］顯示KPC腫瘤模型中的腫瘤體積-時間曲線。 ［圖11A］顯示LL2腫瘤模型中用於體內評估的投予排程。［圖11B］至［圖11C］顯示LL2腫瘤模型中的腫瘤體積-時間曲線。 ［圖12A］顯示LL2-OVA腫瘤模型中用於體內評估的投予排程。［圖12B］至［圖12C］顯示LL2-OVA腫瘤模型中的腫瘤體積-時間曲線。 ［圖13A］顯示皮下EMT6腫瘤模型中用於體內評估的投予排程。［圖13B］至［圖13C］顯示EMT6腫瘤模型中的腫瘤體積-時間曲線。 The following is a brief description of the figures, which are presented for illustrative purposes only and not for limiting the illustrative embodiments disclosed herein. [Figure 1A] through [Figure 1C] show the cell-binding curves of the 9E3 chimera and ABC1931 as detected by FACS. [Figure 2] shows a schematic structure of the anti-FAP-LIGHT fusion protein ABC1930. [Figure 3A] through [Figure 3B] show biolayer interferometry (BLI) sensing maps of immobilized FAP or LTßR ECD proteins using continuously diluted solutions of ABC1930. [Figure 3C] shows a BLI sensing map of immobilized HVEM ECD proteins using continuously diluted solutions of ABC1947 or ABC1930. [Figure 4] Shows the binding of ABC1930 or ABC1947, detected by ELISA, to the coated DcR3 protein. [Figure 5] Shows the binding of ABC1930, detected by ELISA, to the coated human FAP or DPPIV protein. [Figure 6] Shows schematic forms A and D of the fusion protein generated in Example 2.1. SDS-PAGE results indicate that form D, with LIGHTmu1 or LIGHTwt, was correctly assembled, while the chain encoding three tandem hmLIGHT units in form A showed low performance, resulting in poor assembly of the two structures. [Figure 7A] Shows a comparison of the yields of ABC2097, ABC1930, and ABC1947 after purification of protein A. [Figure 7B] shows SDS-PAGE gel images of these proteins. [Figures 8A] through [Figure 8C] show CCL2 production induced by ABC1931, ABC1773, and ABC1930 from both the 3T3 and 3T3-FAP cell lines, respectively. [Figures 9A] through [Figure 9C] show the transactivation of the endogenous LTβR pathway in 3T3 cells by CT26-FAP cells after treatment with ABC1931, ABC1773, or ABC1930, using CCL2 as a substitute readout. [Figure 10A] shows the administration schedule used for in vivo evaluation in a KPC mouse model. [Figures 10B] through 10C show the tumor volume-time curve in the KPC tumor model. [Figure 11A] shows the administration schedule used for in vivo assessment in the LL2 tumor model. [Figures 11B] through 11C show the tumor volume-time curve in the LL2 tumor model. [Figure 12A] shows the administration schedule used for in vivo assessment in the LL2-OVA tumor model. [Figures 12B] through 12C show the tumor volume-time curve in the LL2-OVA tumor model. [Figure 13A] shows the administration schedule used for in vivo assessment in the subcutaneous EMT6 tumor model. Figures 13B to 13C show the tumor volume-time curves in the EMT6 tumor model.

TW202540200A_113146439_SEQL.xmlTW202540200A_113146439_SEQL.xml

Claims (110)

一種經單離抗FAP抗體或其FAP抗原結合片段，其包含： a) i)     重鏈可變區(VH)，其包含 (1)  包含IYGVN (SEQ ID NO: 26)之胺基酸序列的重鏈互補決定區1 (HCDR1)， (2)  包含AIWSGGRKDYX 2LSLKS (SEQ ID NO: 27)之胺基酸序列的重鏈互補決定區2 (HCDR2)，其中X 2係N或S，及 (3)  包含SQDMPGYFDY (SEQ ID NO: 28)之胺基酸序列的重鏈互補決定區3 (HCDR3)，及 ii)    輕鏈可變區(VL)，其包含 (1)  包含KTNQNVDYX 1GNTFMH (SEQ ID NO: 23)之胺基酸序列的輕鏈互補決定區1 (LCDR1)，其中X 1係N或S， (2)  包含LASNLAS (SEQ ID NO: 24)之胺基酸序列的輕鏈互補決定區2 (LCDR2)，及 (3)  包含QQSRNLPYT (SEQ ID NO: 25)之胺基酸序列的輕鏈互補決定區3 (LCDR3)； 或 b) i)     VH區，其包含 (1)  包含TAGMSVG (SEQ ID NO: 32)之胺基酸序列的HCDR1， (2)  包含DIWWDDKKHYNPSLKD (SEQ ID NO: 33)之胺基酸序列的HCDR2， (3)  包含DMIFNFYFDV (SEQ ID NO: 34)之胺基酸序列的HCDR3，及 ii)    VL區，其包含 (1)  包含SASSRVGYMH (SEQ ID NO: 29)之胺基酸序列的LCDR1， (2)  包含DTSKLAS (SEQ ID NO: 30)之胺基酸序列的LCDR2，及 (3)  包含FQGSGYPFT (SEQ ID NO: 31)之胺基酸序列的LCDR3。 A single-chain anti-FAP antibody or its FAP antigen-binding fragment, comprising: a) i) a heavy chain variable region (VH) comprising (1) a heavy chain complement determination region 1 (HCDR1) comprising an amino acid sequence of IYGVN (SEQ ID NO: 26), (2) a heavy chain complement determination region 2 (HCDR2) comprising an amino acid sequence of AIWSGGRKDYX 2 LSLKS (SEQ ID NO: 27), wherein X 2 is N or S, and (3) a heavy chain complement determination region 3 (HCDR3) comprising an amino acid sequence of SQDMPGYFDY (SEQ ID NO: 28), and ii) a light chain variable region (VL) comprising (1) a light chain variable region comprising KTNQNVDYX 1 GNTFMH (SEQ ID NO: 28), wherein X 2 is N or S, and (2) a heavy chain complement determination region 3 (HCDR3) comprising an amino acid sequence of IYGVN (SEQ ID NO: 26), wherein X 2 is N or S, and (3) a light chain variable region comprising (1) a light chain variable region comprising KTNQNVDYX 1 GNTFMH (SEQ ID NO: 28), wherein X 2 is N or S, and (4) a light chain variable region comprising (1) a light chain variable region comprising KTNQNVDYX 1 GNTFMH (SEQ ID NO: 28), wherein X 2 is N or S, and (5) a light chain variable region comprising (1) a light chain variable region comprising KTNQNVDYX 1 GNTFMH (SEQ ID NO: 28), wherein X 2 is N or S, and (6) a light chain variable region comprising (1) a light chain variable region comprising KTNQNVDYX 1 GNTFMH (SEQ ID NO: 28), wherein X 2 is N or S, and (7) a light chain variable region comprising (1) a light chain variable region comprising KTNQNVDYX 1 GNTFMH (SEQ ID NO: 28), wherein X 2 is N 23) Light chain complementarity determining region 1 (LCDR1) of the amino acid sequence, wherein X1 is N or S, (2) Light chain complementarity determining region 2 (LCDR2) of the amino acid sequence of LASSNLAS (SEQ ID NO: 24), and (3) Light chain complementarity determining region 3 (LCDR3) of the amino acid sequence of QQSRNLPYT (SEQ ID NO: 25); or b) i) VH region, which includes (1) HCDR1 of the amino acid sequence of TAGMSVG (SEQ ID NO: 32), (2) HCDR2 of the amino acid sequence of DIWWDDKKHYNPSLKD (SEQ ID NO: 33), (3) HCDR3 of the amino acid sequence of DMIFNFYFDV (SEQ ID NO: 34), and ii) VL region, which includes (1) LCDR1 containing the amino acid sequence SASSRVGYMH (SEQ ID NO: 29), LCDR2 containing the amino acid sequence DTSKLAS (SEQ ID NO: 30), and LCDR3 containing the amino acid sequence FQGSGYPFT (SEQ ID NO: 31). 如請求項1之經單離抗FAP抗體或其FAP抗原結合片段，其中該經單離抗FAP抗體或其FAP抗原結合片段包含： a)     VH區，其包含與如SEQ ID NO: 35所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；及/或VL區，其包含與如SEQ ID NO: 36所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列； b)     VH區，其包含與如SEQ ID NO: 37所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；及/或VL區，其包含與如SEQ ID NO: 38所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；或 c)     VH區，其包含與如SEQ ID NO: 41所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；及/或VL區，其包含與如SEQ ID NO: 42所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列。 The monoclonal antibody against FAP or its FAP antigen-binding fragment as claimed in claim 1, wherein the monoclonal antibody against FAP or its FAP antigen-binding fragment comprises: a) a VH region comprising an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to or identical to the amino acid sequence shown in SEQ ID NO: 35; and/or a VL region comprising an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to or identical to the amino acid sequence shown in SEQ ID NO: 36; b) a VH region comprising an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to or identical to the amino acid sequence shown in SEQ ID NO: 36; b) a VH region comprising an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to or identical to the amino acid sequence shown in SEQ ID NO: 36; The amino acid sequence shown in SEQ ID NO: 37 contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 38; and/or the VL region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 38; or c) the VH region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 41; and/or the VL region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 38; The amino acid sequence shown in 42 contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or an amino acid sequence identical to it. 如請求項1或2之經單離抗FAP抗體或其FAP抗原結合片段，其中該經單離抗FAP抗體或其FAP抗原結合片段包含： a)     重鏈(HC)區，其包含與如SEQ ID NO: 1、4、5、6、8、9、10、11、12、13、14、15、及16所示之胺基酸序列中之任一者包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；及/或 b)     輕鏈(LC)區，其包含與如SEQ ID NO: 2、3、及7所示之胺基酸序列中之任一者包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列。 The monoclonal antibody against FAP or its FAP antigen-binding fragment as claimed in claim 1 or 2, wherein the monoclonal antibody against FAP or its FAP antigen-binding fragment comprises: a) a heavy chain (HC) region comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with or identical to any of the amino acid sequences shown in SEQ ID NO: 1, 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, and 16; and/or b) a light chain (LC) region comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with or identical to any of the amino acid sequences shown in SEQ ID NO: Any of the amino acid sequences shown in 2, 3, and 7 contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or an amino acid sequence identical to it. 如請求項1至3中任一項之經單離抗FAP抗體或其FAP抗原結合片段，其中該經單離抗FAP抗體或其FAP抗原結合片段包含： a)     HC區，其包含與如SEQ ID NO: 1所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；及/或LC區，其包含與如SEQ ID NO: 2所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；或 b)     HC區，其包含與如SEQ ID NO: 4所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；及/或LC區，其包含與如SEQ ID NO: 3所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列； c)     第一重鏈(HC1)區，其包含與如SEQ ID NO: 5所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；第二重鏈(HC2)區，其包含與如SEQ ID NO: 6所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；及/或LC區，其包含與如SEQ ID NO: 3所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列； d)     HC1區，其包含與如SEQ ID NO: 8所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；HC2區，其包含與如SEQ ID NO: 9所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；及/或LC區，其包含與如SEQ ID NO: 7所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列； e)     HC1區，其包含與如SEQ ID NO: 10所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；HC2區，其包含與如SEQ ID NO: 11所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；及/或LC區，其包含與如SEQ ID NO: 3所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列； f)     HC1區，其包含與如SEQ ID NO: 13所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；HC2區，其包含與如SEQ ID NO: 12所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；及/或LC區，其包含與如SEQ ID NO: 3所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列； g)     HC1區，其包含與如SEQ ID NO: 14所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；HC2區，其包含與如SEQ ID NO: 13所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；及/或LC區，其包含與如SEQ ID NO: 3所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；或 h)     HC1區，其包含與如SEQ ID NO: 15所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；HC2區，其包含與如SEQ ID NO: 16所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；及/或LC區，其包含與如SEQ ID NO: 3所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列。 The monoclonal antibody against FAP or its FAP antigen-binding fragment, as claimed in any of claims 1 to 3, comprises: a) an HC region containing an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to or the same as the amino acid sequence shown in SEQ ID NO: 1; and/or an LC region containing an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to or the same as the amino acid sequence shown in SEQ ID NO: 2; or b) an HC region containing an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to or the same as the amino acid sequence shown in SEQ ID NO: 2; The amino acid sequence shown in SEQ ID NO: 4 contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 3; and/or the LC region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 5; c) The first heavy chain (HC1) region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 5; the second heavy chain (HC2) region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 5; The amino acid sequence shown in SEQ ID NO: 6 contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 3; and/or the LC region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 3; d) The HC1 region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 8; the HC2 region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 8; The amino acid sequence shown in SEQ ID NO: 9 contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 7; and/or the LC region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 7; e) the HC1 region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 10; the HC2 region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 10; The amino acid sequence shown in SEQ ID NO: 11 contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 3; and/or the LC region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 3; f) The HC1 region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 13; the HC2 region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 13; The amino acid sequence shown in SEQ ID NO: 12 contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 3; and/or the LC region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 3; g) The HC1 region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 14; the HC2 region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 14; The amino acid sequence shown in SEQ ID NO: 13 contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 3; and/or the LC region, which contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 3; or h) the HC1 region, which contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 15; the HC2 region, which contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 15; The amino acid sequence shown in SEQ ID NO: 3 contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical amino acid sequence; and/or the LC region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical amino acid sequence as shown in SEQ ID NO: 3. 如請求項1至4中任一項之經單離抗FAP抗體或其FAP抗原結合片段，其中該VH區及/或VL區進一步包含人類架構序列。The single-cell anti-FAP antibody or its FAP antigen-binding fragment, as claimed in any of claims 1 to 4, wherein the VH region and/or VL region further comprises a human structural sequence. 如請求項1至5中任一項之經單離抗FAP抗體或其FAP抗原結合片段，其中該VH區及/或VL區進一步包含架構1 (FR1)、架構2 (FR2)、架構3 (FR3)、及/或架構4 (FR4)序列。The single-cell anti-FAP antibody or its FAP antigen-binding fragment, as claimed in any of claims 1 to 5, wherein the VH region and/or VL region further comprises the sequences of structure 1 (FR1), structure 2 (FR2), structure 3 (FR3), and/or structure 4 (FR4). 如請求項1至6中任一項之經單離抗FAP抗體或其FAP抗原結合片段，其包含衍生自免疫球蛋白之可結晶片段(Fc)區。The monoclonal FAP antibody or its FAP antigen-binding fragment, as described in any of claims 1 to 6, includes a crystallizable fragment (Fc) region derived from an immunoglobulin. 如請求項7之經單離抗FAP抗體或其FAP抗原結合片段，其中該Fc片段係衍生自IgG1、IgG2、IgG3、或IgG4，可選地，該Fc片段係衍生自IgG4。For example, the Fc fragment of the monoclonal antibody against FAP or its FAP antigen-binding fragment as claimed in claim 7, wherein the Fc fragment is derived from IgG1, IgG2, IgG3 or IgG4, and optionally, the Fc fragment is derived from IgG4. 如請求項7或8之經單離抗FAP抗體或其FAP抗原結合片段，其中該Fc片段包含突變S228P或LALAPG突變。Such as the monoclonal FAP antibody or its FAP antigen-binding fragment as claimed in claim 7 or 8, wherein the Fc fragment contains the S228P or LALAPG mutation. 如請求項1至9中任一項之經單離抗FAP抗體或其FAP抗原結合片段，其中該抗體係單株抗體。The FAP antibody or its FAP antigen-binding fragment, as described in any of claims 1 to 9, is a monoclonal antibody. 如請求項1至10中任一項之經單離抗FAP抗體或其FAP抗原結合片段，其中該抗體係人源化、人類、或嵌合抗體。The single-cell FAP antibody or its FAP antigen-binding fragment, as requested in any of claims 1 to 10, wherein the antibody is a humanized, human, or chimeric antibody. 如請求項1至11中任一項之經單離抗FAP抗體或其FAP抗原結合片段，其中該抗FAP抗體或FAP抗原結合片段與人類、食蟹獼猴、及小鼠FAP交叉反應。The single anti-FAP antibody or its FAP antigen-binding fragment, as claimed in any of claims 1 to 11, wherein the anti-FAP antibody or FAP antigen-binding fragment cross-reacts with human, cynomolgus monkey, and mouse FAP. 如請求項1至12中任一項之經單離抗FAP抗體或其FAP抗原結合片段，其中該抗體或其片段係Fab、Fab’、F(ab’)2、Fv、scFv、(scFv)2、單鏈抗體分子、雙可變區抗體、單可變區抗體、線性抗體、V區、或由抗體片段形成之多特異性抗體。The FAP antibody or its FAP antigen-binding fragment, as claimed in any of claims 1 to 12, wherein the antibody or its fragment is Fab, Fab’, F(ab’)2, Fv, scFv, (scFv)2, a single-chain antibody molecule, a bivariate antibody, a monovariate antibody, a linear antibody, a V region, or a multispecific antibody formed from an antibody fragment. 如請求項1至13中任一項之經單離抗FAP抗體或其FAP抗原結合片段，其中該經單離抗FAP抗體或其FAP抗原結合片段以不大於20 nM、15 nM、10 nM、或5 nM之解離常數(K D)結合至FAP。 The monoclonal antibody against FAP or its FAP antigen-binding fragment, as claimed in any of claims 1 to 13, binds to FAP with a dissociation constant (K<sub> D </sub>) not greater than 20 nM, 15 nM, 10 nM, or 5 nM. 一種雙特異性抗體或其抗原結合片段，其包含FAP抗原結合部份及第二結合部份，其中該FAP抗原結合部份包含： a) i)     重鏈可變區(VH)，其包含 (1)  包含IYGVN (SEQ ID NO: 26)之胺基酸序列的重鏈互補決定區1 (HCDR1)， (2)  包含AIWSGGRKDYX 2LSLKS (SEQ ID NO: 27)之胺基酸序列的重鏈互補決定區2 (HCDR2)，其中X 2係N或S，及 (3)  包含SQDMPGYFDY (SEQ ID NO: 28)之胺基酸序列的重鏈互補決定區3 (HCDR3)，及 ii)    輕鏈可變區(VL)，其包含 (1)  包含KTNQNVDYX 1GNTFMH (SEQ ID NO: 23)之胺基酸序列的輕鏈互補決定區1 (LCDR1)，其中X 1係N或S， (2)  包含LASNLAS (SEQ ID NO: 24)之胺基酸序列的輕鏈互補決定區2 (LCDR2)，及 (3)  包含QQSRNLPYT (SEQ ID NO: 25)之胺基酸序列的輕鏈互補決定區3 (LCDR3)； 或 b) i)     VH區，其包含 (1)  包含TAGMSVG (SEQ ID NO: 32)之胺基酸序列的HCDR1， (2)  包含DIWWDDKKHYNPSLKD (SEQ ID NO: 33)之胺基酸序列的HCDR2， (3)  包含DMIFNFYFDV (SEQ ID NO: 34)之胺基酸序列的HCDR3，及 ii)    VL區，其包含 (1)  包含SASSRVGYMH (SEQ ID NO: 29)之胺基酸序列的LCDR1， (2)  包含DTSKLAS (SEQ ID NO: 30)之胺基酸序列的LCDR2，及 (3)  包含FQGSGYPFT (SEQ ID NO: 31)之胺基酸序列的LCDR3。 A bispecific antibody or its antigen-binding fragment thereof, comprising an FAP antigen-binding portion and a second binding portion, wherein the FAP antigen-binding portion comprises: a) i) a heavy chain variable region (VH) comprising (1) a heavy chain complementarity-determining region 1 (HCDR1) comprising an amino acid sequence of IYGVN (SEQ ID NO: 26), (2) a heavy chain complementarity-determining region 2 (HCDR2) comprising an amino acid sequence of AIWSGGRKDYX 2 LSLKS (SEQ ID NO: 27), wherein X 2 is N or S, and (3) a heavy chain complementarity-determining region 3 (HCDR3) comprising an amino acid sequence of SQDMPGYFDY (SEQ ID NO: 28), and ii) a light chain variable region (VL) comprising (1) a light chain variable region comprising KTNQNVDYX 2 LSLKS 2 LSLKS 2 LSLKS (SEQ ID NO: 27), wherein X 2 is N or S, and (4) a light chain variable region comprising (5) a light chain variable region comprising (6) a light chain variable region comprising (7) a light chain variable region comprising (8) a light chain variable region comprising (9) a light chain variable region comprising (10 ... 1) a light chain complementarity determining region 1 (LCDR1) of the amino acid sequence of GNTFMH (SEQ ID NO: 23), wherein X1 is N or S, (2) a light chain complementarity determining region 2 (LCDR2) of the amino acid sequence of LASNLAS (SEQ ID NO: 24), and (3) a light chain complementarity determining region 3 (LCDR3) of the amino acid sequence of QQSRNLPYT (SEQ ID NO: 25); or b) i) a VH region comprising (1) HCDR1 of the amino acid sequence of TAGMSVG (SEQ ID NO: 32), (2) HCDR2 of the amino acid sequence of DIWWDDKKHYNPSLKD (SEQ ID NO: 33), (3) HCDR3 of the amino acid sequence of DMIFNFYFDV (SEQ ID NO: 34), and ii) VL region, comprising (1) LCDR1 containing the amino acid sequence SASSRVGYMH (SEQ ID NO: 29), (2) LCDR2 containing the amino acid sequence DTSKLAS (SEQ ID NO: 30), and (3) LCDR3 containing the amino acid sequence FQGSGYPFT (SEQ ID NO: 31). 如請求項15之雙特異性抗體或其抗原結合片段，其中該FAP抗原結合部份包含： a)     VH區，其包含與如SEQ ID NO: 35所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；及/或VL區，其包含與如SEQ ID NO: 36所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列； b)     VH區，其包含與如SEQ ID NO: 37所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；及/或VL區，其包含與如SEQ ID NO: 38所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；或 c)     VH區，其包含與如SEQ ID NO: 41所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；及/或VL區，其包含與如SEQ ID NO: 42所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列。 The bispecific antibody or antigen-binding fragment thereof, as claimed in claim 15, wherein the FAP antigen-binding portion comprises: a) a VH region comprising an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to or the same as the amino acid sequence shown in SEQ ID NO: 35; and/or a VL region comprising an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to or the same as the amino acid sequence shown in SEQ ID NO: 36; b) a VH region comprising an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to or the same as the amino acid sequence shown in SEQ ID NO: 36; The amino acid sequence shown in SEQ ID NO: 37 contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 38; and/or the VL region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 38; or c) the VH region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 41; and/or the VL region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 38; The amino acid sequence shown in 42 contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or an amino acid sequence identical to it. 如請求項15或16之雙特異性抗體或其抗原結合片段，其中該FAP抗原結合部份包含： a)     重鏈(HC)區，其包含與如SEQ ID NO: 1、4、5、6、8、9、10、11、12、13、14、15、及16所示之胺基酸序列中之任一者包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；及/或 b)     輕鏈(LC)區，其包含與如SEQ ID NO: 2、3、及7所示之胺基酸序列中之任一者包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列。 The bispecific antibody or antigen-binding fragment thereof, as claimed in claim 15 or 16, wherein the FAP antigen-binding portion comprises: a) a heavy chain (HC) region comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with or identical to any of the amino acid sequences shown in SEQ ID NO: 1, 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, and 16; and/or b) a light chain (LC) region comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with or identical to any of the amino acid sequences shown in SEQ ID NO: Any of the amino acid sequences shown in 2, 3, and 7 contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or an amino acid sequence identical to it. 如請求項15至17中任一項之雙特異性抗體或其抗原結合片段，其中該FAP抗原結合部份包含： a)     HC區，其包含與如SEQ ID NO: 1所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；及/或LC區，其包含與如SEQ ID NO: 2所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；或 b)     HC區，其包含與如SEQ ID NO: 4所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；及/或LC區，其包含與如SEQ ID NO: 3所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列； c)     第一重鏈(HC1)區，其包含與如SEQ ID NO: 5所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；第二重鏈(HC2)區，其包含與如SEQ ID NO: 6所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；及/或LC區，其包含與如SEQ ID NO: 3所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列； d)     HC1區，其包含與如SEQ ID NO: 8所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；HC2區，其包含與如SEQ ID NO: 9所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；及/或LC區，其包含與如SEQ ID NO: 7所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列； e)     HC1區，其包含與如SEQ ID NO: 10所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；HC2區，其包含與如SEQ ID NO: 11所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；及/或LC區，其包含與如SEQ ID NO: 3所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列； f)     第一HC1區，其包含與如SEQ ID NO: 13所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；HC2區，其包含與如SEQ ID NO: 12所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；及/或LC區，其包含與如SEQ ID NO: 3所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列； g)     HC1區，其包含與如SEQ ID NO: 14所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；HC2區，其包含與如SEQ ID NO: 13所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；及/或LC區，其包含與如SEQ ID NO: 3所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；或 h)     HC1區，其包含與如SEQ ID NO: 15所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；HC2區，其包含與如SEQ ID NO: 16所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列；及/或LC區，其包含與如SEQ ID NO: 3所示之胺基酸序列包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列。 The bispecific antibody or antigen-binding fragment thereof of any one of claims 15 to 17, wherein the FAP antigen-binding portion comprises: a) an HC region comprising an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to or the same as the amino acid sequence shown in SEQ ID NO: 1; and/or an LC region comprising an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to or the same as the amino acid sequence shown in SEQ ID NO: 2; or b) an HC region comprising an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to or the same as the amino acid sequence shown in SEQ ID NO: 2; The amino acid sequence shown in SEQ ID NO: 4 contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 3; and/or the LC region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 5; c) The first heavy chain (HC1) region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 5; the second heavy chain (HC2) region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 5; The amino acid sequence shown in SEQ ID NO: 6 contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 3; and/or the LC region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 3; d) The HC1 region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 8; the HC2 region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 8; The amino acid sequence shown in SEQ ID NO: 9 contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 7; and/or the LC region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 7; e) the HC1 region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 10; the HC2 region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 10; The amino acid sequence shown in SEQ ID NO: 11 contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 3; and/or the LC region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 3; f) The first HC1 region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 13; the HC2 region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 13; The amino acid sequence shown in SEQ ID NO: 12 contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 3; and/or the LC region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 3; g) The HC1 region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 14; the HC2 region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 14; The amino acid sequence shown in SEQ ID NO: 13 contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 3; and/or the LC region, which contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 3; or h) the HC1 region, which contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 15; the HC2 region, which contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical with the amino acid sequence shown in SEQ ID NO: 15; The amino acid sequence shown in SEQ ID NO: 3 contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical amino acid sequence; and/or the LC region contains an amino acid sequence that contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or identical amino acid sequence as shown in SEQ ID NO: 3. 如請求項15至18中任一項之雙特異性抗體或其抗原結合片段，其在該FAP抗原結合部份之N端包含衍生自免疫球蛋白之Fc片段。The bispecific antibody or its antigen-binding fragment of any of claims 15 to 18 shall contain an Fc fragment derived from an immunoglobulin at the N-terminus of the FAP antigen-binding region. 如請求項19之雙特異性抗體或其抗原結合片段，其中該Fc片段係衍生自IgG1、IgG2、IgG3、或IgG4，可選地，該Fc片段係衍生自IgG4。Such as the bispecific antibody or antigen-binding fragment thereof in claim 19, wherein the Fc fragment is derived from IgG1, IgG2, IgG3 or IgG4, and optionally, the Fc fragment is derived from IgG4. 如請求項19或20之雙特異性抗體或其抗原結合片段，其中該Fc片段包含突變S228P或LALAPG突變。Such as bispecific antibodies or antigen-binding fragments thereof as claimed in claims 19 or 20, wherein the Fc fragment contains the S228P or LALAPG mutation. 如請求項15至21中任一項之雙特異性抗體或其抗原結合片段，其中該雙特異性抗體或其抗原結合片段包含Fc片段，且其中該Fc片段包含選自由下列所組成之群組的一或多個修飾：鈕扣(knobs-into-holes)、DDKK、CH3之靜電轉向(electrostatic steering)、DuoBody、SEEDbodies、cFAE、XmAb、Azymetric、及BEAT ®，可選地，該Fc片段包含修飾鈕扣及/或DDKK。 The bispecific antibody or antigen-binding fragment thereof of any of claims 15 to 21, wherein the bispecific antibody or antigen-binding fragment thereof comprises an Fc fragment, and wherein the Fc fragment comprises one or more modifications selected from the group consisting of: knots-into-holes, DDKK, electrostatic steering of CH3, DuoBody, SEEDbodies, cFAE, XmAb, Azymetric, and BEAT® , optionally, the Fc fragment comprises a knot and/or DDKK modification. 如請求項15至22中任一項之雙特異性抗體或其抗原結合片段，其中該第二結合部份係在該FAP抗原結合部份之C端。The bispecific antibody or antigen-binding fragment thereof of any one of claims 15 to 22, wherein the second binding portion is at the C-terminus of the FAP antigen-binding portion. 如請求項19至23中任一項之雙特異性抗體或其抗原結合片段，其中該第二結合部份係可操作地連接至該Fc片段之該C端。The bispecific antibody or antigen-binding fragment thereof of any of claims 19 to 23, wherein the second binding portion is operatively linked to the C-terminus of the Fc fragment. 如請求項19至24中任一項之雙特異性抗體或其抗原結合片段，其中該第二結合部份藉由連接子連接至該Fc片段。Such as a bispecific antibody or antigen-binding fragment thereof of any of claims 19 to 24, wherein the second binding portion is linked to the Fc fragment by a linker. 如請求項15至25中任一項之雙特異性抗體或其抗原結合片段，其中該第二結合部份結合及/或活化第二目標。The bispecific antibody or antigen-binding fragment thereof of any one of claims 15 to 25, wherein the second binding portion binds to and/or activates a second target. 如請求項15至26中任一項之雙特異性抗體或其抗原結合片段，其中該第二結合部份結合及/或活化腫瘤相關細胞受體。The bispecific antibody or antigen-binding fragment thereof of any of claims 15 to 26, wherein the second binding portion binds to and/or activates tumor-associated cell receptors. 如請求項15至27中任一項之雙特異性抗體或其抗原結合片段，其中該第二結合部份包含第一部分及第二部分，其中各部分包含一或多個單元。The bispecific antibody or antigen-binding fragment thereof of any of claims 15 to 27, wherein the second binding portion comprises a first portion and a second portion, wherein each portion comprises one or more units. 如請求項28之雙特異性抗體或其抗原結合片段，其中該一或多個單元包含第一單元、第二單元、及/或第三單元。Such as the bispecific antibody or antigen-binding fragment thereof in claim 28, wherein the one or more units comprise a first unit, a second unit, and/or a third unit. 如請求項28或29之雙特異性抗體或其抗原結合片段，其中該一或多個單元之各者係相同的。Such as the bispecific antibody or antigen-binding fragment thereof in claims 28 or 29, wherein each of the one or more units is identical. 如請求項28或29之雙特異性抗體或其抗原結合片段，其中該一或多個單元係不相同的。Such as bispecific antibodies or antigen-binding fragments thereof in claims 28 or 29, wherein one or more units are distinct. 如請求項15至31中任一項之雙特異性抗體或其抗原結合片段，其中該第二結合部份係腫瘤壞死因子、介白素、淋巴激素、干擾素、群落刺激因子、趨化因子、或生長因子。The bispecific antibody or antigen-binding fragment thereof of any one of claims 15 to 31, wherein the second binding portion is a tumor necrosis factor, interleukin, lymphokine, interferon, community-stimulating factor, chemotherapeutic factor, or growth factor. 如請求項28至31中任一項之雙特異性抗體或其抗原結合片段，其中該一或多個單元之各者獨立地係腫瘤壞死因子、介白素、淋巴激素、干擾素、群落刺激因子、趨化因子、或生長因子。The bispecific antibody or antigen-binding fragment thereof of any one of claims 28 to 31, wherein each of the one or more units is independently a tumor necrosis factor, interleukin, lymphokine, interferon, community-stimulating factor, chemokine, or growth factor. 如請求項29至31中任一項之雙特異性抗體或其抗原結合片段，其中該一或多個單元之各者獨立地包含與 表 10所述之胺基酸序列中之任一者具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%同一性之胺基酸序列。 The bispecific antibody or antigen-binding fragment thereof of any of claims 29 to 31, wherein each of the one or more units independently comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any of the amino acid sequences described in Table 10. 如請求項15至31中任一項之雙特異性抗體或其抗原結合片段，其中該第二結合部份包含第二抗原結合部份或細胞介素部份。The bispecific antibody or antigen-binding fragment thereof of any of claims 15 to 31, wherein the second binding portion comprises a second antigen-binding portion or an intercytokine portion. 如請求項15至35中任一項之雙特異性抗體或其抗原結合片段，其中該第二結合部份包含細胞介素部份。The bispecific antibody or antigen-binding fragment thereof of any one of claims 15 to 35, wherein the second binding portion comprises an intercytokine portion. 如請求項15至36中任一項之雙特異性抗體或其抗原結合片段，其中該細胞介素部份包含第一細胞介素部份及第二細胞介素部份。The bispecific antibody or antigen-binding fragment thereof of any of claims 15 to 36, wherein the intercytokine portion comprises a first intercytokine portion and a second intercytokine portion. 如請求項37之雙特異性抗體或其抗原結合片段，其中該第一細胞介素部份包含第一細胞介素單元。Such as the bispecific antibody or antigen-binding fragment thereof in claim 37, wherein the first intercytokine portion comprises a first intercytokine unit. 如請求項38之雙特異性抗體或其抗原結合片段，其中該第一細胞介素單元包含LIGHT單元或淋巴毒素-β單元。Such as the bispecific antibody or antigen-binding fragment thereof in claim 38, wherein the first intercytokine unit comprises the LIGHT unit or the lymphotoxin-β unit. 如請求項37之雙特異性抗體或其抗原結合片段，其中該第二細胞介素部份包含第二細胞介素單元及第三細胞介素單元。Such as the bispecific antibody or antigen-binding fragment thereof in claim 37, wherein the second intercytokine portion comprises a second intercytokine unit and a third intercytokine unit. 如請求項40之雙特異性抗體或其抗原結合片段，其中該第二細胞介素單元包含LIGHT單元、淋巴毒素-α單元、或淋巴毒素-β單元。Such as the bispecific antibody or antigen-binding fragment thereof in claim 40, wherein the second intercytokine unit comprises the LIGHT unit, the lymphotoxin-α unit, or the lymphotoxin-β unit. 如請求項40之雙特異性抗體或其抗原結合片段，其中該第三細胞介素單元包含LIGHT單元、淋巴毒素-α單元、或淋巴毒素-β單元。Such as the bispecific antibody or antigen-binding fragment thereof in claim 40, wherein the third intercytokine unit comprises the LIGHT unit, the lymphotoxin-α unit, or the lymphotoxin-β unit. 如請求項39至42中任一項之雙特異性抗體或其抗原結合片段，其中該第一細胞介素部份包含第一LIGHT單元，該第二細胞介素單元包含第二LIGHT單元，且該第三細胞介素單元包含第三LIGHT單元。The bispecific antibody or antigen-binding fragment thereof of any of claims 39 to 42, wherein the first intercytokine portion comprises a first LIGHT unit, the second intercytokine unit comprises a second LIGHT unit, and the third intercytokine unit comprises a third LIGHT unit. 如請求項43之雙特異性抗體或其抗原結合片段，其中該第一LIGHT單元、該第二LIGHT單元、及/或該第三LIGHT單元各自獨立地包含 SEQ ID NO: 17或 18所述之胺基酸序列。 The bispecific antibody or antigen-binding fragment thereof, as claimed in claim 43, wherein the first LIGHT unit, the second LIGHT unit, and/or the third LIGHT unit each independently comprises the amino acid sequence described in SEQ ID NO: 17 or 18 . 如請求項39至42中任一項之雙特異性抗體或其抗原結合片段，其中該第一細胞介素部份包含淋巴毒素-β單元，該第二細胞介素單元包含淋巴毒素-α單元，且該第三細胞介素單元包含淋巴毒素-β單元。The bispecific antibody or antigen-binding fragment thereof of any of claims 39 to 42, wherein the first intercytokine portion comprises a lymphotoxin-β unit, the second intercytokine unit comprises a lymphotoxin-α unit, and the third intercytokine unit comprises a lymphotoxin-β unit. 如請求項45之雙特異性抗體或其抗原結合片段，其中該淋巴毒素-β單元包含 SEQ ID NO: 39所述之胺基酸序列，該淋巴毒素-α單元包含 SEQ ID NO: 40所述之胺基酸序列。 The bispecific antibody or antigen-binding fragment thereof, as claimed in claim 45, wherein the lymphotoxin-β unit comprises the amino acid sequence described in SEQ ID NO: 39 , and the lymphotoxin-α unit comprises the amino acid sequence described in SEQ ID NO: 40 . 如請求項15至46中任一項之雙特異性抗體或其抗原結合片段，其中該雙特異性抗體或其抗原結合片段以不大於20 nM、15 nM、10 nM、或5 nM之解離常數(K D)結合至FAP。 The bispecific antibody or antigen-binding fragment thereof of any one of claims 15 to 46, wherein the bispecific antibody or antigen-binding fragment thereof binds to the FAP with a dissociation constant ( KD ) not greater than 20 nM, 15 nM, 10 nM, or 5 nM. 如請求項15至47中任一項之雙特異性抗體或其抗原結合片段，其中該雙特異性抗體或其抗原結合片段以不大於20 nM、15 nM、10 nM、或5 nM之解離常數(K D)結合至LTßR。 The bispecific antibody or antigen-binding fragment thereof of any one of claims 15 to 47, wherein the bispecific antibody or antigen-binding fragment thereof binds to LTßR with a dissociation constant ( KD ) not greater than 20 nM, 15 nM, 10 nM, or 5 nM. 如請求項15至48中任一項之雙特異性抗體或其抗原結合片段，其中該雙特異性抗體或其抗原結合片段幾乎不結合至人類或食蟹獼猴HVEM。The bispecific antibody or antigen-binding fragment thereof of any of claims 15 to 48, wherein the bispecific antibody or antigen-binding fragment thereof binds substantially not to human or cynomolgus monkey HVEM. 如請求項15至49中任一項之雙特異性抗體或其抗原結合片段，其中該雙特異性抗體或其抗原結合片段中之該第二結合部份能夠降低與DcR3之結合親和力。The bispecific antibody or antigen-binding fragment thereof of any one of claims 15 to 49, wherein the second binding portion of the bispecific antibody or antigen-binding fragment thereof is capable of reducing the binding affinity to DcR3. 如請求項15至50中任一項之雙特異性抗體或其抗原結合片段，其中該雙特異性抗體或其抗原結合片段特異性結合至人類FAP，且/或不結合至DPPIV。A bispecific antibody or antigen-binding fragment thereof of any one of claims 15 to 50, wherein the bispecific antibody or antigen-binding fragment thereof specifically binds to human FAP and/or does not bind to DPPIV. 如請求項15之雙特異性抗體或其抗原結合片段，其中該第二結合部份結合及/或活化LTβR、HER2、PDL-1、PD-1、EGFR、VEGFR、VEGF、CCR8、OX-40、418B、血管生成素-2、IL-4Ra、BCMA、Blys、BTNO2、C5、CD122、CD13、CD133、CD137、CD138、CD16a、CD19、CD20、CD22、CD27、CD28、CD3、CD30、CD33、CD38、CD40、CD47、CD-8、CEA、CGPR/CGRPR、CSPGs、CTLA4、CTLA-4、DLL-4、EpCAM、因子IXa、因子X、GITR、GP130、Her3、HSG、ICOS、IGFl、IGFl/2、IGF-lR、IGF2、IGFR、IL-1、IL- 12、IL-12p40、IL-13、IL-l 7A、IL-1~、IL-23、IL-5、IL-6、IL-6R、Lag-3、LAG3、MAG、Met、NgR、NogoA、OMGp、OX40、PDGFR、PSMA、RGMA、RGMB、SARS-CoV-2、Te38、TIM-3、TNF、TNFa、TROP-2、TWEAK、或TRAIL。Such as the bispecific antibody or antigen-binding fragment thereof in claim 15, wherein the second binding portion binds to and/or activates LTβR, HER2, PDL-1, PD-1, EGFR, VEGFR, VEGF, CCR8, OX-40, 418B, angiopoietin-2, IL-4Ra, BCMA, Blys, BTNO2, C5, CD122, CD13, CD133, CD137, CD138, CD16a, CD19, CD2 0, CD22, CD27, CD28, CD3, CD30, CD33, CD38, CD40, CD47, CD-8, CEA, CGPR/CGRPR, CSPGs, CTLA4, CTLA-4, DLL-4, EpCAM, Factor IXa, Factor X, GITR, GP130, Her3, HSG, ICOS, IGF1, IGF1/2, IGF-1R, IGF2, IGFR, IL-1, IL- 12. IL-12p40, IL-13, IL-1 7A, IL-1~, IL-23, IL-5, IL-6, IL-6R, Lag-3, LAG3, MAG, Met, NgR, NogoA, OMGp, OX40, PDGFR, PSMA, RGMA, RGMB, SARS-CoV-2, Te38, TIM-3, TNF, TNFa, TROP-2, TWEAK, or TRAIL. 如請求項52之雙特異性抗體或其抗原結合片段，其中該第二結合部份係第二抗原結合部份。Such as the bispecific antibody or antigen-binding fragment thereof in claim 52, wherein the second binding portion is a second antigen-binding portion. 如請求項52之雙特異性抗體或其抗原結合片段，其中該第二抗原結合部份包含抗LTβR結合部份、抗HER2結合部份、抗PDL-1結合部份、抗PD-1結合部份、抗EGFR結合部份、抗VEGFR結合部份、抗VEGF結合部份、抗CCR8結合部份、抗OX-40結合部份、抗418B結合部份、抗血管生成素-2結合部份、抗IL-4Ra結合部份、抗BCMA結合部份、抗Blys結合部份、抗BTNO2結合部份、抗C5結合部份、抗CD122結合部份、抗CD13結合部份、抗CD133結合部份、抗CD137結合部份、抗CD138結合部份、抗CD16a結合部份、抗CD19結合部份、抗CD20結合部份、抗CD22結合部份、抗CD27結合部份、抗CD28結合部份、抗CD3結合部份、抗CD30結合部份、抗CD33結合部份、抗CD38結合部份、抗CD40結合部份、抗CD47結合部份、抗CD-8結合部份、抗CEA結合部份、抗CGPR/CGRPR結合部份、抗CSPGs結合部份、抗CTLA4結合部份、抗CTLA-4結合部份、抗DLL-4結合部份、抗EpCAM結合部份、抗因子IXa結合部份、抗因子X結合部份、抗GITR結合部份、抗GP130結合部份、抗Her3結合部份、抗HSG結合部份、抗ICOS結合部份、抗IGFl結合部份、抗IGFl/2結合部份、抗IGF-lR結合部份、抗IGF2結合部份、抗IGFR結合部份、抗IL-1結合部份、抗IL-12結合部份、抗IL-12p40結合部份、抗IL-13結合部份、抗IL-l 7A結合部份、抗IL-1~結合部份、抗IL-23結合部份、抗IL-5結合部份、抗IL-6結合部份、抗IL-6R結合部份、抗Lag-3結合部份、抗LAG3結合部份、抗MAG結合部份、抗Met結合部份、抗NgR結合部份、抗NogoA結合部份、抗OMGp結合部份、抗OX40結合部份、抗PDGFR結合部份、抗PSMA結合部份、抗RGMA結合部份、抗RGMB結合部份、抗SARS-CoV-2結合部份、抗Te38結合部份、抗TIM-3結合部份、抗TNF結合部份、抗TNFa結合部份、抗TROP-2結合部份、抗TWEAK結合部份、或抗TRAIL結合部份。Such as the bispecific antibody or its antigen-binding fragment in claim 52, wherein the second antigen-binding portion comprises an anti-LTβR binding portion, an anti-HER2 binding portion, an anti-PDL-1 binding portion, an anti-PD-1 binding portion, an anti-EGFR binding portion, an anti-VEGFR binding portion, an anti-VEGF binding portion, an anti-CCR8 binding portion, an anti-OX-40 binding portion, an anti-418B binding portion, an anti-angiogenic-2 binding portion, and an anti-IL-4Ra binding portion. Partial, anti-BCMA binding region, anti-Blys binding region, anti-BTNO2 binding region, anti-C5 binding region, anti-CD122 binding region, anti-CD13 binding region, anti-CD133 binding region, anti-CD137 binding region, anti-CD138 binding region, anti-CD16a binding region, anti-CD19 binding region, anti-CD20 binding region, anti-CD22 binding region, anti-CD27 binding region, anti-CD28 binding region, anti-CD3 Anti-CD30 binding region, anti-CD33 binding region, anti-CD38 binding region, anti-CD40 binding region, anti-CD47 binding region, anti-CD-8 binding region, anti-CEA binding region, anti-CGPR/CGRPR binding region, anti-CSPGs binding region, anti-CTLA4 binding region, anti-CTLA-4 binding region, anti-DLL-4 binding region, anti-EpCAM binding region, anti-factor IXa binding region, anti-factor X-binding region, anti-GITR binding region, anti-GP130 binding region, anti-Her3 binding region, anti-HSG binding region, anti-ICOS binding region, anti-IGF1 binding region, anti-IGF1/2 binding region, anti-IGF-IR binding region, anti-IGF2 binding region, anti-IGFR binding region, anti-IL-1 binding region, anti-IL-12 binding region, anti-IL-12p40 binding region, anti-IL-13 binding region, anti-IL-1 7A binding region, anti-IL-1 binding region, anti-IL-23 binding region, anti-IL-5 binding region, anti-IL-6 binding region, anti-IL-6R binding region, anti-Lag-3 binding region, anti-LAG3 binding region, anti-MAG binding region, anti-Met binding region, anti-NgR binding region, anti-NogoA binding region, anti-OMGp binding region, anti-OX40 binding region, anti-PDGFR binding region, anti-PSMA binding region, anti-RGMA binding region, anti-RGMB binding region, anti-SARS-CoV-2 binding region, anti-Te38 binding region, anti-TIM-3 binding region, anti-TNF binding region, anti-TNFα binding region, anti-TROP-2 binding region, anti-TWEAK binding region, or anti-TRAIL binding region. 如請求項15至54中任一項之雙特異性抗體或其抗原結合片段，其中相對於比較物雙特異性抗體或其抗原結合片段，該雙特異性抗體或其抗原結合片段展現出降低的與HVEM之結合親和力。The bispecific antibody or antigen-binding fragment thereof of any one of claims 15 to 54, wherein the bispecific antibody or antigen-binding fragment thereof exhibits reduced binding affinity to HVEM relative to the comparison bispecific antibody or antigen-binding fragment thereof. 如請求項15至55中任一項之雙特異性抗體或其抗原結合片段，其中相對於比較物雙特異性抗體或其抗原結合片段，該雙特異性抗體或其抗原結合片段展現出降低的與DcR3之結合親和力。The bispecific antibody or antigen-binding fragment thereof of any one of claims 15 to 55, wherein the bispecific antibody or antigen-binding fragment thereof exhibits reduced binding affinity to DcR3 relative to the comparison bispecific antibody or antigen-binding fragment thereof. 如請求項15至56中任一項之雙特異性抗體或其抗原結合片段，其中相對於比較物雙特異性抗體或其抗原結合片段，該雙特異性抗體或其抗原結合片段展現出降低的與DPPIV之結合親和力。The bispecific antibody or antigen-binding fragment thereof of any of claims 15 to 56, wherein the bispecific antibody or antigen-binding fragment thereof exhibits reduced binding affinity to DPPIV relative to the comparison bispecific antibody or antigen-binding fragment thereof. 如請求項15至57中任一項之雙特異性抗體或其抗原結合片段，其中在使該雙特異性抗體或其抗原結合片段與FAP表現性細胞接觸後，該雙特異性抗體或其抗原結合片段誘導：次級淋巴器官(SLO)之形成、三級淋巴結構(TLS)之形成、免疫細胞之刺激、腫瘤細胞之細胞凋亡、癌症之治療、或其任何組合。The bispecific antibody or antigen-binding fragment thereof of any one of claims 15 to 57, wherein, upon contact of the bispecific antibody or antigen-binding fragment thereof with FAP-expressing cells, the bispecific antibody or antigen-binding fragment thereof induces: the formation of secondary lymphoid organs (SLOs), the formation of tertiary lymphoid structures (TLSs), the stimulation of immune cells, apoptosis of tumor cells, cancer treatment, or any combination thereof. 一種融合蛋白，其中該融合蛋白係包含在如請求項15至58中任一項之雙特異性抗體或其抗原結合片段中。A fusion protein, wherein the fusion protein is contained in a bispecific antibody or an antigen-binding fragment thereof as claimed in any one of claims 15 to 58. 如請求項59之融合蛋白，其中該融合蛋白包含重鏈1 (HC1)區，其包含VH、重鏈恆定域1 (CH1)、及包含重鏈恆定域2 (CH2)及重鏈恆定域3 (CH3)之Fc片段。The fusion protein of claim 59, wherein the fusion protein includes a heavy chain 1 (HC1) region, which includes VH, heavy chain constant 1 (CH1), and an Fc fragment including heavy chain constant 2 (CH2) and heavy chain constant 3 (CH3). 如請求項60之融合蛋白，其中該HC1區包含與如 SEQ ID NO: 1 、4 、5 、8 、10 、13 、14 、及15所示之胺基酸序列中之任一者包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列。 The fusion protein of claim 60, wherein the HC1 region contains an amino acid sequence that is at least 80%, 85 % , 90 % , 91 % , 92 % , 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identical to or the same as any of the amino acid sequences shown in SEQ ID NO: 1, 4, 5, 8, 10, 13, 14, and 15. 如請求項60或61之融合蛋白，其中該HC1區包含與如 SEQ ID NO: 5 、8 、10 、13 、14 、及15所示之胺基酸序列中之任一者包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列。 The fusion protein of claim 60 or 61, wherein the HC1 region contains an amino acid sequence that has at least 80%, 85%, 90 % , 91 %, 92 %, 93 %, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with or is identical to any of the amino acid sequences shown in SEQ ID NO: 5, 8, 10, 13, 14, and 15. 如請求項60之融合蛋白，其中該HC1區之該Fc片段包含與該Fc片段之C端融合的該第二結合部份之一或多個單元。The fusion protein of claim 60, wherein the Fc fragment of the HC1 region includes one or more units of the second binding portion fused to the C-terminus of the Fc fragment. 如請求項63之融合蛋白，其中該HC1區之該Fc片段包含與該HC1 Fc片段之該C端融合的該第二結合部份之第一單元。The fusion protein of claim 63, wherein the Fc fragment of the HC1 region includes a first unit of the second binding portion fused to the C-terminus of the HC1 Fc fragment. 如請求項64之融合蛋白，其中該第二結合部份之該第一單元係藉由第一連接子融合至該Fc單元。As in claim 64, the fusion protein wherein the first unit of the second binding portion is fused to the Fc unit via a first linker. 如請求項60至65中任一項之融合蛋白，其中該第二結合部份係細胞介素部份，且其中該細胞介素部份包含第一細胞介素單元。The fusion protein of any of claims 60 to 65, wherein the second binding portion is an intercytokine portion, and wherein the intercytokine portion comprises a first intercytokine unit. 如請求項46之融合蛋白，其中該第一細胞介素單元包含LIGHT單元或淋巴毒素β單元。The fusion protein, such as in claim 46, wherein the first intercytokine unit comprises a LIGHT unit or a lymphotoxin β unit. 如請求項63至66中任一項之融合蛋白，其中該第一單元包含與 表 10所述之胺基酸序列中之任一者具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%同一性或與其同一之胺基酸序列。 The fusion protein of any of claims 63 to 66, wherein the first unit comprises an amino acid sequence having at least 80%, 85 % , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with or the same as any of the amino acid sequences described in Table 10. 如請求項63至68中任一項之融合蛋白，其中該單元包含與SEQ ID NO: 17或SEQ ID NO: 39具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%同一性或與其同一之胺基酸序列。The fusion protein of any one of claims 63 to 68, wherein the unit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with or the same as that of SEQ ID NO: 17 or SEQ ID NO: 39. 如請求項59之融合蛋白，其中該融合蛋白包含重鏈2 (HC2)區，其包含VH、CH1、及包含CH1及CH3之Fc片段。The fusion protein of claim 59, wherein the fusion protein includes a heavy chain 2 (HC2) region comprising VH, CH1, and an Fc fragment comprising CH1 and CH3. 如請求項70之融合蛋白，其中該HC2區包含與如 SEQ ID NO: 1 、4 、6 、9 、11 、12 、13 、及16所示之胺基酸序列中之任一者包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列。 The fusion protein of claim 70, wherein the HC2 region contains an amino acid sequence that has at least 80%, 85 % , 90 % , 91% , 92 % , 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with or is identical to any of the amino acid sequences shown in SEQ ID NO: 1, 4, 6, 9, 11, 12, 13, and 16. 如請求項70或71之融合蛋白，其中該HC2區包含與如 SEQ ID NO: 6 、9 、11 、12 、13 、及16所示之胺基酸序列中之任一者包含至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%序列同一性或與其同一之胺基酸序列。 The fusion protein of claim 70 or 71, wherein the HC2 region contains an amino acid sequence that has at least 80%, 85%, 90 % , 91 %, 92 %, 93 %, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with or is identical to any of the amino acid sequences shown in SEQ ID NO: 6, 9, 11, 12, 13, and 16. 如請求項70之融合蛋白，其中該HC2區之該Fc片段包含與該HC2 Fc片段之C端融合的該第二結合部份之一或多個單元。The fusion protein of claim 70, wherein the Fc fragment of the HC2 region includes one or more units of the second binding portion fused to the C-terminus of the HC2 Fc fragment. 如請求項70之融合蛋白，其中該HC2區之該Fc片段包含該第二結合部份之第二單元及第三單元。The fusion protein of claim 70, wherein the Fc fragment of the HC2 region includes the second and third units of the second binding portion. 如請求項74之融合蛋白，其中該第二結合部份之該第二單元係融合至該HC2區之該Fc片段。The fusion protein of claim 74, wherein the second unit of the second binding portion is fused to the Fc fragment of the HC2 region. 如請求項75之融合蛋白，其中該第二結合部份之該第三單元係融合至該第二結合部份之該第二單元。As in claim 75, the fusion protein wherein the third unit of the second binding portion is fused to the second unit of the second binding portion. 如請求項76之融合蛋白，其中該第二結合部份之該第二單元係藉由第二連接子融合至該HC2區之該Fc片段，且該第三單元係藉由第三連接子融合至該第二結合部份之該第二單元。As in claim 76, the fusion protein wherein the second unit of the second binding portion is fused to the Fc fragment of the HC2 region via a second linker, and the third unit is fused to the second unit of the second binding portion via a third linker. 如請求項77之融合蛋白，其中該第二結合部份單元之該第二單元及該第三單元係串聯連接。The fusion protein of claim 77, wherein the second unit and the third unit of the second binding portion are connected in series. 如請求項73至78之融合蛋白，其中該第二結合部份係細胞介素部份，且其中該細胞介素部份包含第二細胞介素單元及第三細胞介素單元。The fusion protein of claims 73 to 78, wherein the second binding portion is an intercytokine portion, and wherein the intercytokine portion comprises a second intercytokine unit and a third intercytokine unit. 如請求項79之融合蛋白，其中該第二細胞介素單元包含LIGHT單元、淋巴毒素α單元、或淋巴毒素β單元。The fusion protein of claim 79, wherein the second intercytokine unit comprises a LIGHT unit, a lymphotoxin α unit, or a lymphotoxin β unit. 如請求項79或80之融合蛋白，其中該第三細胞介素單元包含LIGHT單元、淋巴毒素α單元、或淋巴毒素β單元。The fusion protein, such as in claims 79 or 80, wherein the third intercytokine unit comprises the LIGHT unit, the lymphotoxin α unit, or the lymphotoxin β unit. 如請求項79至81中任一項之融合蛋白，其中該第二細胞介素單元包含LIGHT單元，且該第三細胞介素單元包含LIGHT單元。The fusion protein of any of claims 79 to 81, wherein the second intercytokine unit contains a LIGHT unit and the third intercytokine unit contains a LIGHT unit. 如請求項79至81中任一項之融合蛋白，其中該第二細胞介素單元包含淋巴毒素α單元，且該第三細胞介素單元包含淋巴毒素β單元。The fusion protein of any of claims 79 to 81, wherein the second intercytokine unit comprises a lymphotoxin α unit and the third intercytokine unit comprises a lymphotoxin β unit. 如請求項74至83中任一項之融合蛋白，其中該第二單元包含與 表 10所述之胺基酸序列中之任一者具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%同一性或與其同一之胺基酸序列。 The fusion protein of any of claims 74 to 83, wherein the second unit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with or the same as any of the amino acid sequences described in Table 10. 如請求項84之融合蛋白，其中該第二單元包含與 SEQ ID NO: 17或 SEQ ID NO: 40具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%同一性或與其同一之胺基酸序列。 The fusion protein of claim 84, wherein the second unit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with or the same as that of SEQ ID NO : 17 or SEQ ID NO: 40 . 如請求項74至85中任一項之融合蛋白，其中該第三單元包含與 表 10所述之胺基酸序列中之任一者具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%同一性或與其同一之胺基酸序列。 The fusion protein of any of claims 74 to 85, wherein the third unit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with or the same as any of the amino acid sequences described in Table 10. 如請求項86之融合蛋白，其中該第三單元包含與 SEQ ID NO: 17或 SEQ ID NO: 39具有至少80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%同一性或與其同一之胺基酸序列。 The fusion protein of claim 86, wherein the third unit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with or the same as that of SEQ ID NO : 17 or SEQ ID NO: 39 . 如請求項65或77之融合蛋白，其中該融合蛋白包含一或多個連接子。The fusion protein, such as in claim 65 or 77, comprises one or more linkers. 如請求項88之融合蛋白，其中該一或多個連接子之各者獨立地包含VH-CHl連接子ASTKGPSVFPLAPS；VL-CL連接子RTVAAPSVFIFPPS (SEQ ID NO: 91)；CH2-CH3連接子ISKAKGQPREPQ (SEQ ID NO: 92)；IgM尾部連接子KSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 93)；GGGGSGGGGSGGGGSGGGGT (SEQ ID NO: 94)；G；或(GGGGS) n(SEQ ID NO: 95)，其中n=l、2、3、4、5、6、7、8、9、或10。 The fusion protein of claim 88, wherein each of the one or more linkers independently comprises the VH-CH1 linker ASTKGPSVFPLAPS; the VL-CL linker RTVAAPSVFIFPPS (SEQ ID NO: 91); the CH2-CH3 linker ISKAKGQPREPQ (SEQ ID NO: 92); the IgM tail linker KSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 93); GGGGSGGGGSGGGGSGGGGT (SEQ ID NO: 94); G; or (GGGGS) n (SEQ ID NO: 95), wherein n = 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. 如請求項63至69中任一項之融合蛋白，其中該HC1區包含一或多個異二聚化修飾。The fusion protein of any of claims 63 to 69, wherein the HC1 region contains one or more heterodimerization modifications. 如請求項90之融合蛋白，其中該一或多個異二聚化修飾係鈕(knob)修飾或孔(hole)修飾。For example, in the fusion protein of claim 90, the one or more heterodimerization modifications are button modifications or hole modifications. 如請求項90或91之融合蛋白，其中該一或多個異二聚化修飾係孔修飾。For example, the fusion protein in request item 90 or 91, wherein the one or more heterodimerization modifications are pore modifications. 如請求項70至87中任一項之融合蛋白，其中該HC2區包含一或多個異二聚化修飾。The fusion protein of any of claims 70 to 87, wherein the HC2 region contains one or more heterodimerization modifications. 如請求項93之融合蛋白，其中該一或多個異二聚化修飾係鈕(knob)修飾或孔(hole)修飾。For example, in the fusion protein of claim 93, the one or more heterodimerization modifications are button modifications or hole modifications. 如請求項93或94之融合蛋白，其中該一或多個異二聚化修飾係鈕修飾。For example, the fusion protein in claim 93 or 94, wherein the one or more heterodimerization modifications are button modifications. 一種經單離多核苷酸，其包含編碼下列之一或多個核苷酸序列： a)     如請求項1至14中任一項之經單離抗FAP抗體或其FAP抗原結合片段中之任一者、 b)     如請求項15至58中任一項之雙特異性抗體中之任一者、或 c)     如請求項59至95中任一項之融合蛋白中之任一者。 A monoclonal polynucleotide comprising encoding one or more of the following nucleotide sequences: a) any of the monoclonal anti-FAP antibodies or FAP antigen-binding fragments thereof as described in any of claims 1 to 14; b) any of the bispecific antibodies as described in any of claims 15 to 58; or c) any of the fusion proteins as described in any of claims 59 to 95. 一種構築體，其包含如請求項96之多核苷酸。A structure comprising the polynucleotides as requested in claim 96. 一種抗體表現系統，其包含如請求項97之構築體或包含如請求項96之多核苷酸的載體。An antibody expression system comprising a construct as claimed in claim 97 or a carrier comprising a polynucleotide as claimed in claim 96. 如請求項98之抗體表現系統，其中該抗體表現系統係細胞表現系統。Such as the antibody expression system in claim 98, wherein the antibody expression system is a cell expression system. 一種用於產生該抗FAP抗體或其FAP抗原結合片段、雙特異性抗體或其抗原結合片段、或融合蛋白之方法，該方法包含：在適合表現該該抗FAP抗體或其FAP抗原結合片段、雙特異性抗體或其抗原結合片段、或融合蛋白的條件下，使用如請求項98或99之抗體表現系統。A method for producing the anti-FAP antibody or its FAP antigen-binding fragment, a bispecific antibody or its antigen-binding fragment, or a fusion protein, the method comprising: using an antibody expression system as described in claims 98 or 99, under conditions suitable for expressing the anti-FAP antibody or its FAP antigen-binding fragment, a bispecific antibody or its antigen-binding fragment, or a fusion protein. 一種醫藥組成物，其中該醫藥組成物包含： a)     如請求項1至14中任一項之經單離抗FAP抗體或其FAP抗原結合片段中之任一者、 b)     如請求項15至58中任一項之雙特異性抗體中之任一者、或 c)     如請求項59至95中任一項之融合蛋白中之任一者；及 醫藥上可接受之載劑。 A pharmaceutical composition comprising: a) any of the monoclonal FAP antibody or its FAP antigen-binding fragment as claimed in any of claims 1 to 14; b) any of the bispecific antibodies as claimed in any of claims 15 to 58; or c) any of the fusion proteins as claimed in any of claims 59 to 95; and a pharmaceutically acceptable delivery system. 一種套組，其中該套組包含： a)     如請求項1至14中任一項之經單離抗FAP抗體或其FAP抗原結合片段中之任一者、 b)     如請求項15至58中任一項之雙特異性抗體中之任一者、或 c)     如請求項59至95中任一項之融合蛋白中之任一者、 d)     如請求項96之經單離多核苷酸、或 e)     如請求項97之構築體。 A kit comprising: a) any of the isolated FAP antibody or its FAP antigen-binding fragment as claimed in any of claims 1 to 14; b) any of the bispecific antibodies as claimed in any of claims 15 to 58; c) any of the fusion proteins as claimed in any of claims 59 to 95; d) an isolated polynucleotide as claimed in claim 96; e) a construct as claimed in claim 97. 一種該抗FAP抗體或其FAP抗原結合片段、雙特異性抗體或其抗原結合片段、或融合蛋白於製造用於預防、診斷、或治療疾病、病症、或病況之治療劑的用途，其中： a)     該抗FAP抗體或其FAP抗原結合片段係如請求項1至14中任一項之經單離抗FAP抗體或其FAP抗原結合片段中之任一者， b)     該雙特異性抗體或其抗原結合片段係如請求項15至58中任一項之雙特異性抗體中之任一者，或 c)     該融合蛋白係如請求項59至95中任一項之融合蛋白中之任一者。 Use of the anti-FAP antibody or its FAP antigen-binding fragment, bispecific antibody or its antigen-binding fragment, or fusion protein in the manufacture of a therapeutic agent for the prevention, diagnosis, or treatment of a disease, condition, or illness, wherein: a) the anti-FAP antibody or its FAP antigen-binding fragment is any of the isolated anti-FAP antibody or its FAP antigen-binding fragment as claimed in any of claims 1 to 14; b) the bispecific antibody or its antigen-binding fragment is any of the bispecific antibodies as claimed in any of claims 15 to 58; or c) the fusion protein is any of the fusion proteins as claimed in any of claims 59 to 95. 如請求項103之用途，其中該疾病、病症、或病況包含腫瘤疾病。For the purposes of request item 103, the disease, condition, or illness includes tumors. 如請求項103或104之用途，其中至少一個腫瘤細胞表現FAP。For the purposes of claims 103 or 104, at least one tumor cell exhibits FAP. 如請求項104至105中任一項之用途，其中該等腫瘤疾病係實體腫瘤。The use of any of claims 104 to 105, wherein the tumor disease is a solid tumor. 如請求項104至106中任一項之用途，其中該等腫瘤疾病包含胃癌、肝癌、肺癌、結腸直腸癌、乳癌、前列腺癌、皮膚癌、骨癌、多發性骨髓瘤、神經膠質瘤、卵巢癌、胰臟癌、子宮頸癌、甲狀腺癌、喉癌、急性骨髓性白血病、慢性骨髓性白血病、急性淋巴母細胞白血病、慢性淋巴母細胞白血病、腦腫瘤、神經母細胞瘤、視網膜母細胞瘤、頭頸癌、唾液腺癌、及淋巴瘤。For the purposes described in any of claims 104 to 106, the tumor diseases include stomach cancer, liver cancer, lung cancer, colorectal cancer, breast cancer, prostate cancer, skin cancer, bone cancer, multiple myeloma, glioma, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and lymphoma. 一種用於治療有需要之對象的方法，其包含向該對象投予治療有效量的： a)     如請求項1至14中任一項之經單離抗FAP抗體或其FAP抗原結合片段中之任一者、 b)     如請求項15至58中任一項之雙特異性抗體中之任一者、 c)     如請求項59至95中任一項之融合蛋白中之任一者、或 d)     如請求項101之醫藥組成物。 A method for treating a subject in need, comprising administering to the subject a therapeutically effective amount of: a) any of the monoclonal anti-FAP antibodies or FAP antigen-binding fragments thereof as claimed in any of claims 1 to 14; b) any of the bispecific antibodies as claimed in any of claims 15 to 58; c) any of the fusion proteins as claimed in any of claims 59 to 95; or d) a pharmaceutical composition as claimed in claim 101. 一種用於降低腫瘤生長速率或腫瘤細胞數目之方法，其包含使腫瘤細胞與有效量的下列接觸： a)     如請求項1至14中任一項之經單離抗FAP抗體或其FAP抗原結合片段中之任一者、 b)     如請求項15至58中任一項之雙特異性抗體中之任一者、 c)     如請求項59至95中任一項之融合蛋白中之任一者、或 d)     如請求項101之醫藥組成物。 A method for reducing tumor growth rate or tumor cell count, comprising contacting tumor cells with an effective amount of: a) any of the monoclonal anti-FAP antibodies or FAP antigen-binding fragments thereof as claimed in any of claims 1 to 14; b) any of the bispecific antibodies as claimed in any of claims 15 to 58; c) any of the fusion proteins as claimed in any of claims 59 to 95; or d) a pharmaceutical composition as claimed in claim 101. 一種殺滅腫瘤細胞之方法，其包含使腫瘤細胞與有效量的下列接觸： a)     如請求項1至14中任一項之經單離抗FAP抗體或其FAP抗原結合片段中之任一者、 b)     如請求項15至58中任一項之雙特異性抗體中之任一者、 c)     如請求項59至95中任一項之融合蛋白中之任一者、或 d)     如請求項101之醫藥組成物。 A method for killing tumor cells, comprising contacting the tumor cells with an effective amount of any of the following: a) any of the monoclonal anti-FAP antibodies or FAP antigen-binding fragments thereof as claimed in any of claims 1 to 14; b) any of the bispecific antibodies as claimed in any of claims 15 to 58; c) any of the fusion proteins as claimed in any of claims 59 to 95; or d) a pharmaceutical composition as claimed in claim 101.
TW113146439A 2023-12-01 2024-11-29 Anti-fap-light fusion protein and use thereof TW202540200A (en)

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