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WO2025210181A1 - Protéines de liaison à l'antigène ciblant un peptide kk-lc-1 à restriction hla - Google Patents

Protéines de liaison à l'antigène ciblant un peptide kk-lc-1 à restriction hla

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Publication number
WO2025210181A1
WO2025210181A1 PCT/EP2025/059178 EP2025059178W WO2025210181A1 WO 2025210181 A1 WO2025210181 A1 WO 2025210181A1 EP 2025059178 W EP2025059178 W EP 2025059178W WO 2025210181 A1 WO2025210181 A1 WO 2025210181A1
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WO
WIPO (PCT)
Prior art keywords
seq
antigen binding
amino acid
acid sequence
binding protein
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/EP2025/059178
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English (en)
Inventor
Fabian Bert SCHEIFELE
Anna Maria SOBIERAJ
Nagjie Laila ALIJAJ
Stephanie JUNGMICHEL
Philip KNOBEL
Leonardo Borras
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Cdr-Life AG
CDR Life AG
Original Assignee
Cdr-Life AG
CDR Life AG
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Filing date
Publication date
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Publication of WO2025210181A1 publication Critical patent/WO2025210181A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3023—Lung
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2833—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against MHC-molecules, e.g. HLA-molecules
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/32—Immunoglobulins specific features characterized by aspects of specificity or valency specific for a neo-epitope on a complex, e.g. antibody-antigen or ligand-receptor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • This disclosure relates to novel antigen binding proteins that bind to tumor peptide-MHC (pMHC) complexes with high specificity, having favorable properties for therapeutic purposes.
  • pMHC binding proteins may be incorporated into CARs or further comprise a CD3 targeting moiety which provide efficient T-cell mediated cancer cell killing despite very low levels of pMHC on the cell surface.
  • Kita-Kyushu lung cancer antigen-1 (KK-LC-1) is a cancer/testis antigen, a class of cancer antigens that are characterized by their spontaneous immunogenicity and unique expression pattern. Cancer/testis antigens are normally expressed only in the germ cells of the normal human testis and placenta but are also activated in tumor cells.
  • the KK-LC-1 peptide NTDNNLAVY (SEQ ID NO: 68) is displayed on major histocompatibility complexes (MHCs) in a variety of cancer cell types, including those of high unmet medical need.
  • Peptide-MHC complexes derived from intracellular tumor associated antigens (TAAs), such as KK-LC-1 derived NTDNNLAVY (SEQ ID NO: 68), represent a large repertoire of novel targets for immunotherapy. They have been traditionally targeted by TCR-engineered T cells or soluble recombinant T-cell receptors (TCRs) fused to an anti-CD3 fragment.
  • TAAs tumor associated antigens
  • TCRs soluble recombinant T-cell receptors
  • Naturally occurring cancer reactive TCRs typically exhibit low binding affinities for their pMHC targets.
  • Kita-Kyushu lung cancer antigen-1 (KK-LC-1/CT83) peptide is presented on HLA-A*01 :01 is a promising tumor target as its expression is restricted to tumors and it is broadly expressed in many cancers including gastric, lung, breast, and cervical cancer, including many unmet need tumors.
  • targeting the KK-LC-1 presenting MHCs is challenging due to the high levels of similar off-target peptides presented in healthy tissues.
  • the present invention relates to antigen binding proteins which specifically bind to a major histocompatibility complex (MHC)-displayed KK-LC-1 peptide NTDNNLAVY (SEQ ID NO: 68) which was inter alia identified on NSCLC samples.
  • MHC major histocompatibility complex
  • the antigen binding proteins advantageously possess high specificity for said MHC-displayed NTDNNLAVY (SEQ ID NO: 68), with binding being dictated by at least 6 amino acids within NTDNNLAVY (SEQ ID NO: 68).
  • An adapted screening process and intense specificity profiling throughout lead optimization was required to generate safe antigen binding proteins with drug like properties.
  • Bispecific antigen binding proteins targeting NTDNNLAVY (SEQ ID NO: 68) and CD3 showed high potency with no off- tumor activity.
  • HLA-restricted off-target peptide shows a significant reduction in affinity to an HLA-restricted off-target peptide, compared to HLA-restricted NTDNNLAVY (SEQ ID NO: 68).
  • Off-target peptides expressed in healthy tissues may pose a cross-reactivity risk.
  • said HLA- restricted off-target peptide is derived from the gene product of ICE1, DSG3, PTS, KDM7A, FUS, PTPRC, ROBO1, TBL3, CHTOP, DMXL2, FAP, GPAM, PDE10A, VAV1, ZBTB40 and/or ZNF430.
  • TBL3 derived peptide (TADHNLLLY; SEQ ID NO: 78) has been identified as having similarity to the KK-LC1 target peptide NTDNNLAVY (SEQ ID NO: 68) and being highly expressed among many healthy tissues and immune cells.
  • the disclosure provides an antigen binding protein which specifically binds to a major histocompatibility complex (MHC)-displayed NTDNNLAVY (SEQ ID NO: 68), wherein the antigen binding protein has at least about a 10-fold reduction in affinity to MHC-displayed TADHNLLLY (SEQ ID NO: 78), relative to the affinity to MHC-displayed NTDNNLAVY (SEQ ID NO: 68).
  • MHC major histocompatibility complex
  • the antigen binding protein has at least a 50-fold decrease in affinity (e.g., an at least 75-fold, 80-fold, 90-fold, 100-fold, 120-fold, 150-fold, or at least a 200-fold decrease in affinity) for the MHC-displayed peptide TADHNLLLY (SEQ ID NO: 62), relative to MHC-displayed NTDNNLAVY (SEQ ID NO: 68), as determined by SPR.
  • affinity e.g., an at least 75-fold, 80-fold, 90-fold, 100-fold, 120-fold, 150-fold, or at least a 200-fold decrease in affinity
  • the antigen binding protein has at least about a 10- fold reduction in affinity to two or more MHC-displayed YTDNWLAVY (SEQ ID NO: 73), ETDNNIVVY (SEQ ID NO: 74), PTDENLARY (SEQ ID NO: 75), NTDNLLTEY (SEQ ID NO: 76), NSDSNLTTY (SEQ ID NO: 77), NSDNNTIFV (SEQ ID NO: 79), NLDKNLIKY (SEQ ID NO: 80), QLDNQLDAY (SEQ ID NO: 81), SVDSNLFVY (SEQ ID NO: 82), SADNNIVLY (SEQ ID NO: 83), GGDNQLLLY (SEQ ID NO: 85), NLDQSLAHY (SEQ ID NO: 86), FTDNQILLK (SEQ ID NO: 87), GADRNLLVY (SEQ ID NO: 88), relative to the affinity to MHC-displayed NTDN
  • the disclosure provides an antigen binding protein which specifically binds to a major histocompatibility complex (MHC)-displayed NTDNNLAVY (SEQ ID NO: 68), wherein the antigen binding protein has a binding specificity dictated by at least 4 amino acid residues in MHC-displayed NTDNNLAVY (SEQ ID NO: 68).
  • MHC major histocompatibility complex
  • said 4 amino acid residues are not anchor residues.
  • said EC50-value is increased by at least 20-fold, at least 50-fold, at least 100-fold, or by at least 1000-fold.
  • said substitutions are N5A, L6A, A7S, V8A, N5R, L6R, A7R, V8R, N5D, L6D, A7D or V8D of NTDNNLAVY (SEQ ID NO: 68).
  • the antigen binding protein has at least a 50-fold decrease in affinity (e.g., a 75-fold, 80-fold, 90-fold, 100-fold, 120-fold, 150-fold, or 200- fold decrease in affinity) for the MHC-displayed peptides selected from the group consisting of YTDNWLAVY (SEQ ID NO: 73), ETDNNIVVY (SEQ ID NO: 74), NTDNLLTEY (SEQ ID NO: 76), NSDSNLTTY (SEQ ID NO: 77), TADHNLLLY (SEQ ID NO: 78), NSDNNTIFV (SEQ ID NO: 79), NLDKNLIKY (SEQ ID NO: 80), QLDNQLDAY (SEQ ID NO: 81), SVDSNLFVY (SEQ ID NO: 82), SADNNIVLY (SEQ ID NO: 83), GGDNQLLLY (SEQ ID NO: 85), NLDQSLAHY (SEQ ID NO: 73),
  • the antigen binding protein has at least a 50-fold decrease in affinity (e.g., a 75-fold, 80-fold, 90-fold, 100-fold, 120-fold, 150-fold, or 200- fold decrease in affinity) for the MHC-displayed peptide TADHNLLLY (SEQ ID NO: 78), relative to MHC-displayed NTDNNLAVY (SEQ ID NO: 68), as determined by SPR.
  • affinity e.g., a 75-fold, 80-fold, 90-fold, 100-fold, 120-fold, 150-fold, or 200- fold decrease in affinity
  • the MHC is of HLA supertype A*01, in particular HLA-A*01:01.
  • the antigen binding protein is not a T Cell Receptor (TCR).
  • the antigen binding protein comprises:
  • an antibody heavy chain variable (VH) domain comprising an HCDR1 amino acid sequence of XiSYYMC, wherein Xi is R or S (SEQ ID NO: 140), an HCDR2 amino acid sequence of CIYAGSSGSTYYASWAKG (SEQ ID NO: 141), and an HCDR3 amino acid sequence of GAGYGNX2GHSL, wherein X2 is D or G (SEQ ID NO: 142); and/or (ii) an antibody light chain variable (VL) domain comprising an LCDR1 amino acid sequence of X3ASX4NIYNSLA, wherein X3 is Q or R, and X4 is E or K (SEQ ID NO: 143), an LCDR2 amino acid sequence of GASNLES (SEQ ID NO: 144), and an LCDR3 amino acid sequence of QXsTYYGHDNXeGGA, wherein X5 is S or A, and X 6 is V or I (SEQ ID NO: 145).
  • the antigen binding protein comprises: an antibody heavy chain variable (VH) domain comprising an HCDR1 amino acid sequence of SSYYYMC (SEQ ID NO: 155), an HCDR2 amino acid sequence of CIYAGSSGSTYYASWAKG (SEQ ID NO: 156), and an HCDR3 amino acid sequence of GAGYGNGGHSL (SEQ ID NO: 157); and an antibody light chain variable (VL) domain comprising an LCDR1 amino acid sequence of RASKNIYNSLA (SEQ ID NO: 152), an LCDR2 amino acid sequence of GASNLES (SEQ ID NO: 153), and an LCDR3 amino acid sequence of QATYYGHDNIGGA (SEQ ID NO: 154).
  • VH antibody heavy chain variable
  • the antigen binding protein comprises:
  • an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 160
  • an antibody light chain variable (VL) domain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 161; or
  • the antigen binding comprises: (a) an antibody heavy chain variable (VH) domain comprising an HCDR1 amino acid sequence of SSYYYMC (SEQ ID NO: 25), an HCDR2 amino acid sequence of CIYAGSSGSTYYASWAKG (SEQ ID NO: 26), and an HCDR3 amino acid sequence of GAGYGNDGHSL (SEQ ID NO: 27); and (b) an antibody light chain variable (VL) domain comprising an LCDR1 amino acid sequence of QASENIYNSLA (SEQ ID NO: 28), an LCDR2 amino acid sequence of GASNLES (SEQ ID NO: 29), and an LCDR3 amino acid sequence of QSTYYGHDNVGGA (SEQ ID NO: 30).
  • VH antibody heavy chain variable
  • (iii) protein has a binding specificity dictated by at least 4 amino acid residues in said MHC-displayed NTDNNLAVY (SEQ ID NO: 68). such that the EC50- value of Granzyme B (GrzB) expression is increased by at least a 10-fold when one of said at least 4 amino acid residues is substituted by N5A, L6A, A7S, V8A, N5R, L6R, A7R, V8R, N5D, L6D, A7D or V8D of NTDNNLAVY (SEQ ID NO: 68).
  • GrzB Granzyme B
  • said variant of the VL domain comprises the LCDR1 amino acid sequence of RASKNIYNSLA (SEQ ID NO: 146), the LCDR2 amino acid sequence of GASNLES (SEQ ID NO: 147), and the LCDR3 amino acid sequence of QATYYGHDNVGGA (SEQ ID NO: 148).
  • the VH and VL are joined with an amino acid linker.
  • the amino acid linker comprises the amino acid sequence GGGGS (SEQ ID NO: 2), GGGGSGGGGS (SEQ ID NO: 3), GGGGSGGGGSGGGGS (SEQ ID NO: 4), GGGGS GGGGS GGGGS GGGGS (SEQ ID NO: 5), GGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 6), or GGGGSGGGGSGGGGSGGGGAS (SEQ ID NO: 7).
  • the antigen binding protein is or comprises a scFv with an amino acid sequence that is at least about 79%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 37; SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168 and SEQ ID NO: 169.
  • the antigen binding protein is chemically or biologically modified.
  • a glutamine (Q) or glutamate (E) at position 1 of the light chain and/or heavy chain is replaced by pyroglutamate (pE).
  • the antigen binding protein is linked to or combined with a functional entity such as a detectable label, a therapeutic agent or a PK modifying moiety.
  • the disclosure provides an immune cell expressing the CAR described herein, in particular wherein the immune cell is a T cell.
  • the disclosure provides an antibody drug conjugate (ADC) comprising the antigen binding protein described herein.
  • ADC antibody drug conjugate
  • the disclosure provides a multispecific antigen binding protein comprising the antigen binding protein described herein.
  • the multispecific antigen binding protein of is bispecific or trispecific.
  • the multispecific antigen binding protein comprises: (1) a first antigen binding domain, said first antigen binding domain being the antigen binding protein described herein; and (2) an immune cell binding domain.
  • the immune cell binding domain targets CD3, TCRa, TCRP, the a/ T Cell Receptor, CD 16a, NKG2D, CD94/NKG2C, NKp30, NKp46, CD89, CD64, and CD32a on the surface of an immune cell, in particular a human immune cell.
  • the immune cell binding domain is an antibody, more particularly a CD3 binding domain, a CD 16a binding domain or BMA031 or a variant thereof.
  • the CD3 binding domain comprises: (a) an antibody heavy chain variable (VH) domain comprising an HCDR1 amino acid sequence of STYAMN (SEQ ID NO: 46) an HCDR2 amino acid sequence of RIRSKYNNYATYYADSVKG (SEQ ID NO: 47), and an HCDR3 amino acid sequence of HGNFGDSYVSWFAY (SEQ ID NO: 48); and (b) an antibody light chain variable (VL) domain comprising an LCDR1 amino acid sequence of GSSTGAVTTSNYAN (SEQ ID NO: 49), an LCDR2 amino acid sequence of GTNKRAP (SEQ ID NO: 50), and an LCDR3 amino acid sequence of ALWYSNHWV (SEQ ID NO: 51).
  • VH antibody heavy chain variable domain comprising an HCDR1 amino acid sequence of STYAMN (SEQ ID NO: 46) an HCDR2 amino acid sequence of RIRSKYNNYATYYADSVKG (SEQ ID NO: 47), and an HCDR3 amino acid sequence of HGN
  • the VH domain comprises an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 43
  • the VL domain comprises an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 44.
  • the multispecific antigen binding protein comprises or consisting of a heavy chain domain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98, 99%% or 100% identical to the amino acid sequence of SEQ ID NO: 43, and a light chain domain comprising or consisting of an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 45.
  • the CD3 binding domain comprises: (a) an antibody heavy chain variable (VH) domain comprising an HCDR1 amino acid sequence of STYAMN (SEQ ID NO: 56) an HCDR2 amino acid sequence of RIRSKFNNYATYYADSVKG (SEQ ID NO: 57), and an HCDR3 amino acid sequence of HGNFGDSYVSWFAY (SEQ ID NO: 58); and (b) an antibody light chain variable (VL) domain comprising an LCDR1 amino acid sequence of RSSTGAVTTSNYAN (SEQ ID NO: 59), an LCDR2 amino acid sequence of GTNKRAP (SEQ ID NO: 60), and an LCDR3 amino acid sequence of ALWYSNHWV (SEQ ID NO: 61).
  • VH antibody heavy chain variable domain comprising an HCDR1 amino acid sequence of STYAMN (SEQ ID NO: 56) an HCDR2 amino acid sequence of RIRSKFNNYATYYADSVKG (SEQ ID NO: 57), and an HCDR3 amino acid
  • the VH domain comprises an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 54
  • the VL domain comprises an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 55.
  • a HC comprising or consisting of an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 40; and a LC comprising or consisting of an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 41; or
  • a HC comprising or consisting of an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 40; and a LC comprising or consisting of an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 42; or
  • a HC comprising or consisting of an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 52; and a LC comprising or consisting of an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 53.
  • the multispecific antigen binding protein is a Fab- scFv, comprising
  • the scFv of SEQ ID NO: 167 being linked to the C-terminus of the HC of SEQ ID NO: 52; and a LC of SEQ ID NO: 53; or [085] the scFv of SEQ ID NO: 168, being linked to the C-terminus of the HC of SEQ ID NO: 40; and a LC of SEQ ID NO: 41; or
  • the multispecific antigen binding protein is a Fab- scFv, comprising or consisting of:
  • a HC comprising or consisting of an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 62; and a LC comprising or consisting of an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 63; or
  • a HC comprising or consisting of an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 64; and a LC comprising or consisting of an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 65; or
  • a HC comprising or consisting of an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 66; and a LC comprising or consisting of an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 67.
  • the multispecific antigen binding protein further comprises a second antigen binding domain.
  • said second antigen binding domain binds to MHC-displayed NTDNNLAVY (SEQ ID NO: 68).
  • said second antigen binding domain has at least about a 10-fold reduction in affinity to one or more of MHC-displayed YTDNWLAVY (SEQ ID NO: 73), ETDNNIVVY (SEQ ID NO: 74), NTDNLLTEY (SEQ ID NO: 76), NSDSNLTTY (SEQ ID NO: 77), NSDNNTIFV (SEQ ID NO: 79), NLDKNLIKY (SEQ ID NO: 80), QLDNQLDAY (SEQ ID NO: 81), SVDSNLFVY (SEQ ID NO: 82), SADNNIVLY (SEQ ID NO: 83), GGDNQLLLY (SEQ ID NO: 85), NLDQSLAHY (SEQ ID NO: 86), FTDNQILLK (SEQ ID NO: 87), or GADRNLLVY (SEQ ID NO: 88), relative to the affinity to MHC-displayed NTDNNLAVY (SEQ ID NO: 73), ETDNNIVVY
  • an antibody heavy chain variable (VH) domain comprising an HCDR1 amino acid sequence of XiSYYMC, wherein Xi is R or S (SEQ ID NO: 140), an HCDR2 amino acid sequence of CIYAGSSGSTYYASWAKG (SEQ ID NO: 141), and an HCDR3 amino acid sequence of GAGYGNX2GHSL, wherein X2 is D or G (SEQ ID NO: 142); and/or (b) an antibody light chain variable (VL) domain comprising an LCDR1 amino acid sequence of X3ASX4NIYNSLA, wherein X3 is Q or R, and X4 is E or K (SEQ ID NO: 143), an LCDR2 amino acid sequence of GASNLES (SEQ ID NO: 144), and an LCDR3 amino acid sequence of QXsTYYGHDNXeGGA, wherein X5 is S or A, and X 6 is V or I (SEQ ID NO: 145);
  • said second antigen binding domain comprises:
  • the second antigen binding protein comprises:
  • an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 160
  • an antibody light chain variable (VL) domain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 161;
  • VH antibody heavy chain variable domain
  • VL antibody light chain variable domain
  • an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 35
  • an antibody light chain variable (VL) domain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 36;
  • an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 162
  • an antibody light chain variable (VL) domain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 163.
  • the multispecific antigen binding protein is a variant comprising 1, 2, 3, 4, 5, or 6 substitutions in any one or more of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 or LFR4, wherein said variant
  • (i) has at least 50-fold decrease in affinity (e.g., a 75-fold, 80-fold, 90- fold, 100-fold, 120-fold, 150-fold, or 200-fold decrease in affinity) for the MHC-displayed peptides selected from the group consisting of YTDNWLAVY (SEQ ID NO: 73), ETDNNIVVY (SEQ ID NO: 74), NTDNLLTEY (SEQ ID NO: 76), NSDSNLTTY (SEQ ID NO: 77), NSDNNTIFV (SEQ ID NO: 79), NLDKNLIKY (SEQ ID NO: 80), QLDNQLDAY (SEQ ID NO: 81), SVDSNLFVY (SEQ ID NO: 82), SADNNIVLY (SEQ ID NO: 83), GGDNQLLLY (SEQ ID NO: 85), NLDQSLAHY (SEQ ID NO: 86), FTDNQILLK (SEQ ID NO: 87), GA
  • (iii) protein has a binding specificity dictated by at least 4 amino acid residues in said MHC-displayed NTDNNLAVY (SEQ ID NO: 68) such that the EC50- value of Granzyme B (GrzB) expression is increased by at least a 10-fold when each of said at least 4 amino acid residues is substituted is substituted by N5A, L6A, A7S, V8A, N5R, L6R, A7R, V8R, N5D, L6D, A7D or V8D of NTDNNLAVY (SEQ ID NO: 68).
  • GrzB Granzyme B
  • said variant of the VH domain comprises the HCDR1 amino acid sequence of SSYYYMC (SEQ ID NO: 155), the HCDR2 amino acid sequence of CIYAGSSGSTYYASWAKG (SEQ ID NO: 156), and the HCDR3 amino acid sequence of GAGYGNGGHSL (SEQ ID NO: 157)
  • said variant of the VH domain comprises the HCDR1 amino acid sequence of SSYYYMC (SEQ ID NO: 25), the HCDR2 amino acid sequence of CIYAGSSGSTYYASWAKG (SEQ ID NO: 26), and the HCDR3 amino acid sequence of GAGYGNDGHSL (SEQ ID NO: 27).
  • said variant of the VH domain comprises the HCDR1 amino acid sequence of RSYYYMC (SEQ ID NO: 149), the HCDR2 amino acid sequence of CIYAGSSGSTYYASWAKG (SEQ ID NO: 150), and the HCDR3 amino acid sequence of GAGYGNDGHSL (SEQ ID NO: 151).
  • said variant of the VL domain comprises the LCDR1 amino acid sequence of RASKNIYNSLA (SEQ ID NO: 152), the LCDR2 amino acid sequence of GASNLES (SEQ ID NO: 153), and the LCDR3 amino acid sequence of QATYYGHDNIGGA (SEQ ID NO: 154).
  • said variant of the VL domain comprises the LCDR1 amino acid sequence of QASENIYNSLA (SEQ ID NO: 28), the LCDR2 amino acid sequence of GASNLES (SEQ ID NO: 29), and the LCDR3 amino acid sequence of QSTYYGHDNVGGA (SEQ ID NO: 30).
  • said variant of the VL domain comprises the LCDR1 amino acid sequence of RASKNIYNSLA (SEQ ID NO: 146), the LCDR2 amino acid sequence of GASNLES (SEQ ID NO: 147), and the LCDR3 amino acid sequence of QATYYGHDNVGGA (SEQ ID NO: 148).
  • said second antigen binding domain is the antigen binding protein described herein.
  • said second antigen binding domain is identical to the first antigen binding domain.
  • any one or more of the first and second antigen binding domain and the immune cell binding domain is or comprises a full-length immunoglobulin or an antibody fragment.
  • the antibody fragment is or comprises a Fab fragment, a F(ab’)2 fragment, a Fab’ fragment, an Fv fragment, a single chain variable fragment (scFv), and a single domain antibody fragment.
  • the first antigen binding domain and/or the second antigen binding domain is or comprises a scFv.
  • the VH and VL of the first and/or the second antigen binding domain are joined with an amino acid linker.
  • the amino acid linker comprises (GGGGS)n (SEQ ID NO: 1), wherein n is an integer between 1 and 5.
  • the amino acid linker comprises the amino acid sequence GGGGS (SEQ ID NO: 2), GGGGSGGGGS (SEQ ID NO: 3), GGGGSGGGGSGGGGS (SEQ ID NO: 4), or GGGGS GGGGS GGGGS GGGGS (SEQ ID NO: 5).
  • the amino acid linker comprises GGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 6), or
  • GGGGSGGGGSGGGGSGGGGAS (SEQ ID NO: 7).
  • the multispecific antigen binding protein is or comprises a (scFv)s, tribody, Fab?, Fabs, Fab4, or scFv-Fab-scFv (Fab-scFv?).
  • the immune cell binding domain is a Fab fragment, said Fab fragment comprising (1) a heavy chain comprising a CHI domain and the VH, and (2) a light chain comprising a CL domain and the VL.
  • the multispecific antigen binding protein comprises the Fab fragment as defined herein.
  • the CHI domain comprises the amino acid sequence EPKSC (SEQ ID NO: 17) of an antibody hinge region.
  • the first antigen binding domain is operably linked to the C-terminus of the heavy chain or the N-terminus of the heavy chain of the Fab fragment.
  • the second antigen binding domain is operably linked to the C-terminus of the light chain or the N-terminus of the light chain of the Fab fragment.
  • the first antigen binding domain is or comprises an scFv being linked to the C-terminus of the Fab domain heavy chain and the second antigen binding domain comprises an scFv being linked to the C-terminus of the Fab domain light chain; b) the first antigen binding domain is or comprises an scFv being linked to the N-terminus of the Fab domain heavy chain and the second antigen binding domain comprises an scFv being linked to the N-terminus of the Fab domain light chain; c) the first antigen binding domain is or comprises an scFv being linked to the N-terminus of the Fab domain heavy chain and the second antigen binding domain comprises an scFv being linked to the C-terminus of the Fab domain light chain; or d) the first antigen binding domain is or comprises an scFv being linked to the C-terminus of the Fab domain heavy chain and the second antigen binding domain comprises an scFv being linked to the N- terminus of the Fab domain light chain; or d)
  • the scFv is linked to the Fab domain with an amino acid linker.
  • the amino acid linker comprises (GGGGS)n (SEQ ID NO: 1), wherein n is an integer between 1 and 5.
  • the multispecific antigen binding protein does not comprise an Fc domain.
  • the multispecific antigen binding protein is a bispecific Fab-scFv2 comprising:
  • a HC comprising or consisting of an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 178; and a LC comprising or consisting of an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 179.
  • the light chain and/or heavy chain comprises an N-terminal and/or C-terminal truncation of 1, 2, 3, 4, or 5 amino acids.
  • the light chain comprises an N-terminal truncation of 1 or 2 amino acids.
  • the multispecific antigen binding protein comprises a pyroglutamate (pE) at position 1 instead of glutamine (Q) or glutamate (E) of the light chain and/or heavy chain.
  • pE pyroglutamate
  • the multispecific antigen binding protein comprises a pyroglutamate (pE) at position 1 instead of glutamine (Q) or glutamate (E) of the light chain.
  • pE pyroglutamate
  • the disclosure provides a nucleic acid encoding the antigen binding protein described herein, the CAR described herein, or the multispecific antigen binding protein described herein, in particular an isolated nucleic acid.
  • the disclosure provides a vector comprising the nucleic acid described herein.
  • the vector may be an expression vector or a viral vector.
  • the disclosure provides a host cell population comprising the vector of the nucleic acid described herein or the nucleic acid described herein.
  • the disclosure provides a method of manufacturing the antigen binding protein described herein, the ADC described herein, or the multispecific antigen binding protein described herein, comprising the steps of: (i) cultivating the host cell population described herein under conditions allowing expression of the antigen binding protein or the multispecific antigen binding protein; (ii) recovering the antigen binding protein or the multispecific antigen binding protein; and optionally (iii) further purifying and/or modifying and/or formulating the antigen binding protein or the multispecific antigen binding protein.
  • the antigen binding protein described herein is for use in diagnostics.
  • the disclosure provides a kit comprising the antigen binding protein described herein, the CAR described herein, the ADC described herein, or the multispecific antigen binding protein described herein.
  • the disclosure provides the antigen binding protein described herein, the CAR described herein, the immune cell described herein, the ADC described herein, the multispecific antigen binding protein described herein, the nucleic acid described herein, the vector described herein, or the host cell described herein, for use in a method for inhibiting growth or proliferation of cancer cells.
  • the disclosure provides the antigen binding protein described herein, the CAR described herein, the immune cell described herein, the ADC described herein, the multispecific antigen binding protein described herein, the nucleic acid described herein, the vector described herein, or the host cell described herein , for use in a method of redirecting a T cell to a KK-LC-1 -expressing cancer cell.
  • the disclosure provides the antigen binding protein described herein, the CAR described herein, the immune cell described herein, the ADC described herein, the multispecific antigen binding protein described herein, the nucleic acid described herein, the vector described herein, or the host cell described herein , for use as medicament.
  • the disclosure provides the antigen binding protein described herein, the CAR described herein, the immune cell described herein, the ADC described herein, the multispecific antigen binding protein described herein, the nucleic acid described herein, the vector described herein, or the host cell described herein , for use in the treatment of a disease, in particular cancer.
  • the disclosure provides the antigen binding protein described herein, the CAR described herein, the immune cell described herein, the ADC described herein, the multispecific antigen binding protein described herein, the nucleic acid described herein, the vector described herein, or the host cell described herein , for use in the treatment of cancer, wherein the cancer is a solid tumor and/or a hematological tumor, optionally wherein the cancer is selected from cervical cancer, pancreatic cancer, cervical cancer, esophageal cancer, gastric cancer such as gastric adenocarcinoma, lung cancer such as adenocarcinoma, lung squamous cancer or Non-small cell lung cancer (NSCLC) (e.g., non-squamous NSCLC), or breast cancer such as triple-negative breast cancer.
  • NSCLC Non-small cell lung cancer
  • the disclosure provides for the use of the antigen binding protein described herein, the CAR described herein, the immune cell described herein, the ADC described herein, the multispecific antigen binding protein described herein, the nucleic acid described herein, the vector described herein, the host cell described herein , or the pharmaceutical composition described herein, in the manufacture of a medicament.
  • the disclosure provides the antigen binding protein described herein, the CAR described herein, the immune cell described herein, the ADC described herein, the multispecific antigen binding protein described herein, the nucleic acid described herein, the vector described herein, the host cell described herein, or the pharmaceutical composition described herein, for use as medicament.
  • the disclosure provides for the use of the antigen binding protein described herein, the CAR described herein, the immune cell described herein, the ADC described herein, the multispecific antigen binding protein described herein, the nucleic acid described herein, the vector described herein, the host cell described herein or the pharmaceutical composition described herein, in the treatment of a disease, in particular cancer.
  • the cancer is a solid tumor and/or a hematological tumor, optionally wherein the cancer is selected from cervical cancer, pancreatic cancer, cervical cancer, esophageal cancer, gastric cancer such as gastric adenocarcinoma, lung cancer (such as adenocarcinoma, lung squamous cancer or Non-small cell lung cancer (NSCLC) (e.g., non-squamous NSCLC)), or breast cancer such as triple-negative breast cancer.
  • gastric cancer such as gastric adenocarcinoma
  • lung cancer such as adenocarcinoma, lung squamous cancer or Non-small cell lung cancer (NSCLC) (e.g., non-squamous NSCLC)
  • NSCLC Non-small cell lung cancer
  • breast cancer such as triple-negative breast cancer.
  • the disclosure provides the peptide TADHNLLLY (SEQ ID NO: 78) for use as off-target peptide in the generation or selection of an antigen binding molecule targeting an (MHC)-displayed antigen.
  • said antigen is a peptide derived from KK-LC-1, in particular NTDNNLAVY (SEQ ID NO: 68).
  • Fig. 1 depicts HLA-A*01 :01/KK-LC-l identification in non-small cell lung cancer (NSCLC) biopsies.
  • Target quantification was performed by immunoprecipitation followed by mass spectrometry analysis of the tumor and normal adjacent tissue (NAT).
  • Fig. 2 depicts expression levels of off-target peptides with sequence similarity to the KK-LC-1 target peptide in healthy tissues, as determined by immunopeptidomic analysis. Numbers refer to normalized intensity score: a value of 1000 refers to 0.1% of the total peptide signal intensity from the entire sample.
  • Fig. 3 depicts expression levels of off-target peptides with sequence similarity to the KK-LC-1 target peptide in healthy immune cells, as determined by immunopeptidomic analysis. Numbers refer to normalized intensity score: a value of 1000 refers to 0.1% of the total peptide signal intensity from the entire sample.
  • Fig. 4 depicts interferon gamma (IFNy) release as a measure of T cell activation upon treatment with M2883 of antigen presenting T2A1 cells.
  • T2A1 cells were loaded with KK-LC-1 target peptide and physiologically relevant off-target peptides displaying high sequence identity to KK-LC-1 and confirmed presentation in healthy tissues. ULOQ - upper limit of quantification.
  • Fig. 5 depicts Granzyme B (GrzB) release upon treatment of antigen presenting T2A1 cells with M2883 in the presence of PBMCs.
  • T2A1 cells were loaded with KK-LC-1 target peptide and mutated variants thereof with alanine, arginine and aspartate substitutions at each amino acid position. Alanine at position seven and aspartate at position three were substituted with serine.
  • Fig. 6 depicts in vitro cytotoxicity of M2883 in KK-LC-1 -positive HLA-
  • A*01 :01 -positive cancer cells NCI-H1703 and EKVX and KK-LC-1 -negative HLA-
  • A*01 :01 -positive cancer cells SK-MEL-30 and PC-3.
  • FIG. 7 depicts in vitro safety of M2883 in (Fig. 7A) HLA-A*01 flpositive primary cells HCMEC 147 (Human Cardiac Microvascular Endothelial Cells), HBSMC 297 (Human Bronchial Smooth Muscle Cells), HSAEpC_645 (Human Small Airway Epithelial Cells) and HPMEC 770 (Human Pulmonary Microvascular Endothelial Cells), and Fig. 7B) human cardiac fibroblasts (HCF 722), normal human lung fibroblasts (NHLF 19232) and human aortic smooth muscle cells (HAoSMC_173).
  • HCMEC 147 Human Cardiac Microvascular Endothelial Cells
  • HBSMC 297 Human Bronchial Smooth Muscle Cells
  • HSAEpC_645 Human Small Airway Epithelial Cells
  • HPMEC 770 Human Pulmonary Microvascular Endothelial Cells
  • Fig. 7B human cardiac fibro
  • Human pulmonary fibroblasts (HPF 646) served as an HLA-A*01 :01 -negative, KK-LC- 1-negative control.
  • HLA-A*01 :01 -positive and KK-LC-1 -positive cancer cell line NCI- H1703 served as a positive control.
  • FIG. 8 depicts in vitro safety of M2883 and M3903 in HLA-A*01 flpositive primary human lung fibroblasts (NHLF 19232) and human aortic smooth muscle cells (HAoSMC_173).
  • Fig. 9 depicts the quality of M3904 drug substance produced using a stable CHO pool and 14-day fed-batch process at a 10 L scale. Protein purity was assessed by size-exclusion chromatography (SEC), cation-exchange chromatography (CEX) and hydrophobic interaction chromatography (HIC). MP - main peak.
  • SEC size-exclusion chromatography
  • CEX cation-exchange chromatography
  • HIC hydrophobic interaction chromatography
  • FIG. 10 depicts in vitro cytotoxicity of M3904, M3748 and M3864 in KK-
  • Fig. 11 depicts the release of fFNy, IL-2, IL-6, TNFa, Granzyme B and IL-10 following treatment with M3904.
  • the HLA-A*01 :01 -positive and KK-LC-1- positive cancer cell line NCI-H1703 was incubated with PBMCs and varying concentrations of M3904. Cytokine levels were quantified in the culture supernatants.
  • Fig. 12 depicts T cell activation following treatment with M3904.
  • HLA-A*01 :01 -positive and KK-LC-1 -positive cancer cell line NCI-H1703 was incubated with PBMCs and varying concentrations of M3904.
  • T cell activation markers CD69, CD25 and T cell proliferation marker Ki67 were quantified on CD3+, CD4+ and CD8+ T cells.
  • HLA-A*01:01 -positive human primary cells HLA-A*01 :01 -positive, KK-LC-1 -positive control cancer cell line NCI-H1703 served as a positive control.
  • Fig. 14 depicts a molecular binding profile of M3904.
  • Fig 14A depicts a heat map representing the binding of M3903, a monovalent variant of M3904, to a series of peptides, each containing a single amino acid substitution, as determined by SPR.
  • the x-axis represents the position of the substituted amino acid within the peptide sequence, while the y-axis denotes the specific amino acid residue introduced at each position.
  • Color intensity corresponds to binding affinity, which was normalized to the native KK-LC-1 peptide affinity value, with a gradient from white (no binding) to black (200 % and higher binding than to KK-LC-1), as indicated by the accompanying color scale.
  • Fig 14B depicts a sequence logo generated based on the X-scan analysis representing the binding preferences of M3903 and M3904.
  • the height of each letter at a given position corresponds to the relative binding contribution of that amino acid, with larger letters indicating stronger binding preferences.
  • the x-axis represents the position of the amino acid within the peptide sequence, while the y-axis denotes the information content, reflecting sequence conservation and binding specificity.
  • Fig. 15 depicts Granzyme B (GrzB) release upon treatment of antigen presenting T2A1 cells with M3904 in the presence of PBMCs.
  • T2A1 cells were loaded with KK-LC-1 target peptide as positive control and potential off-target peptides posing cross-reactivity risk.
  • Fig. 16 depicts the developability properties of M3904, M3748 and
  • the antigen binding proteins disclosed herein bind to an epitope of CD3 that is conserved among the CD3 antigens from different species, such as non-human primates (e.g., cynomolgus monkeys) or rodents (e.g., mice, rats).
  • the antigen binding proteins are not cross-reactive with a CD3 antigen from rodents (e.g., mouse or rat) or minipigs.
  • the term “antigen binding protein” refers to a protein that specifically binds to or is immunologically reactive with an antigen or epitope.
  • the term “antigen binding protein” includes “antibody” or more particularly “antibody fragment”, but also non-immunoglobulin-based binding proteins.
  • Non-limiting examples of non- immunoglobulin-based binding proteins include DARPins, affimers, monobodies, anticalins, fynomers, and affibodies.
  • Non-immunoglobulin-based binding proteins are described in further detail in Simeon et al. (Protein Cell. 2018. 9(1): 3-14) and Olaleye et al. (Biomolecules. 2021. 11(12): 1791), each ofwhich is incorporated herein by reference.
  • antibody refers to an immunoglobulin molecule or immunoglobulin derived molecule that specifically binds to, or is immunologically reactive with an antigen or epitope, and includes both polyclonal and monoclonal antibodies, as well as functional antibody fragments, including but not limited to fragment antigen-binding (Fab) fragments, F(ab’)2 fragments, Fv fragments, recombinant IgG (rlgG) fragments, single chain variable fragments (scFv) and single domain antibodies (e.g., sdAb, sdFv, nanobody, VHH fragments).
  • the antibody may thus be a single domain antibody or comprise at least one variable light and at least one variable heavy chain.
  • the at least one variable light and at least one variable heavy chain are displayed as a single polypeptide chain.
  • the term “antibody” or “antigen binding protein” includes germline derived antibodies.
  • the term “antibody” or “antigen binding protein” includes genetically engineered or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, heteroconjugate antibodies (e.g., bispecific antibodies, diabodies, triabodies, tetrabodies, tandem di-scFv, tandem tri-scFv) and the like. Unless otherwise stated, the term “antibody” should be understood to encompass functional antibody fragments thereof. [0189] In certain embodiments, the antigen binding protein is not a T cell receptor (TCR), including but not limited to, a soluble TCR.
  • TCR T cell receptor
  • Non-limiting examples of multivalent antigen binding proteins include full-length immunoglobulins, F(ab’)2 fragments, bis-scFv (or tandem scFv or BiTE), DART, diabodies, scDb, DVD-Ig, IgG-scFab, scFab-Fc- scFab, IgG-scFv, scFv- Fc, scFv-fc-scFv, Fv2-Fc, FynomABs, quadroma, CrossMab, DuoBody, triabodies and tetrabodies.
  • the multivalent antigen binding protein is bivalent, i.e., two binding sites are present.
  • the multivalent antigen binding protein is bispecific, i.e., the antigen binding protein is directed against two different targets or two different target sites on one target molecule.
  • the multivalent antigen binding protein includes more than two, e.g., three or four different binding sites for three or four, respectively, different antigens.
  • Such antigen binding protein is multivalent and multispecific, in particular tri- or tetra- specific, respectively.
  • the antigen binding proteins are multispecific
  • the immune cell target antigen is CD3.
  • the multispecific antigen binding protein is a bispecific targeting CD3 andMHC-displayedNTDNNLAVY (SEQ ID NO: 68).
  • said bispecific antigen binding protein is monovalent for CD3 and bivalent for MHC-displayed NTDNNLAVY (SEQ ID NO: 68).
  • the term “monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., antibodies that bind to the same epitope and/or are identical in sequence.
  • a population of polyclonal antibodies in contrast, will bind to multiple epitopes and comprises antibodies of different sequences.
  • a monoclonal antibody preparation may or may not comprise to a minor extent variant antibodies, e.g., due to naturally occurring mutations.
  • variants may e.g., be generated through posttranslational modifications, such as clipping at the N-terminal or C- terminal end of the light and/or heavy chain, or pyroglutamate formation on the N terminus of the polypeptide chain (see e.g., Liu YD, et al. J Biol Chem. 2011 Apr 1;286(13): 11211- 7).
  • posttranslational modifications such as clipping at the N-terminal or C- terminal end of the light and/or heavy chain, or pyroglutamate formation on the N terminus of the polypeptide chain (see e.g., Liu YD, et al. J Biol Chem. 2011 Apr 1;286(13): 11211- 7).
  • the percentage of variants in the mixture varies, and may involve substantially all antibodies produced, or a very low percentage.
  • a “single-chain variable fragment” is an antigen binding protein comprising a heavy chain variable domain (VH) linked to a light chain variable domain (VL).
  • VH and VL domains of the scFv are linked via any appropriate art recognized linker.
  • linkers include, but are not limited to, repeated GGGGS (SEQ ID NO: 2) amino acid sequences or variants thereof.
  • the scFv is generally free of antibody constant domain regions, although an scFv of the disclosure may be linked or attached to antibody constant domain regions (e.g., antibody Fc domain) to alter various properties of the scFv, including, but not limited to, increased serum or tissue half-life.
  • an scFv generally has a molecular weight of about 25 kDa and a hydrodynamic radius of about 2.5 nm.
  • a “Fab fragment” or “Fab” or “Fab domain” is an antibody fragment comprising a light chain fragment comprising a variable light (VL) domain and a constant domain of the light chain (CL), and variable heavy (VH) domain and a first constant domain (CHI) of the heavy chain.
  • a F(ab’)2 comprises two antigen-binding regions joined at the hinge through disulfides.
  • single domain antibody is an antigen binding protein comprising a single heavy chain variable domain derived from the species of the Camelidae family, which includes camels, llama, alpaca.
  • a VHH generally has a molecular weight of about 15 kDa.
  • the antigen binding proteins of the disclosure may comprise one or more linkers for linking the domains of the antigen binding protein (e.g., linking a VH and VL to form a scFv, or linking multiple binding domains to form a multispecific antigen binding protein).
  • linkers for linking the domains of the antigen binding protein e.g., linking a VH and VL to form a scFv, or linking multiple binding domains to form a multispecific antigen binding protein.
  • linkers include glycine polymers (Gly) n ; glycineserine polymers (Gly n Ser) n , where n is an integer of at least one, two, three, four, five, six, seven, or eight; glycine-alanine polymers; alanine-serine polymers; and other flexible linkers known in the art.
  • Glycine and glycine-serine polymers are relatively unstructured, and therefore may be able to serve as a neutral tether between domains of fusion proteins such as the antigen binding proteins described herein. Glycine accesses significantly more phi- psi space than other small side chain amino acids and is much less restricted than residues with longer side chains (Scheraga, Rev. Computational Chem. 1 : 1173-142 (1992)).
  • design of an antigen binding protein in particular embodiments can include linkers that are all or partially flexible, such that the linker can include flexible linker stretches as well as one or more stretches that confer less flexibility to provide a desired structure.
  • Linker sequences can however be chosen to resemble natural linker sequences, for example, using the amino acid stretches corresponding to the beginning of human CHI and CK sequences or amino acid stretches corresponding to the lower portion of the hinge region of human IgG.
  • the design of the peptide linkers connecting VL and VH domains in the scFv moi eties are flexible linkers generally composed of small, non-polar or polar residues such as, e.g., Gly, Ser and Thr.
  • a particularly exemplary linker connecting the variable domains of the scFv moieties is the (Gly4Ser)4 linker (SEQ ID NO: 4), where 4 is the exemplary number of repeats of the motif.
  • Linkers connecting the scFv antigen binding proteins to the Fab domain are also envisioned.
  • the scFv antigen binding proteins are linked to the CHI and CL domains of the Fab with a Gly-Ser linker.
  • the linker comprises the amino acid sequence GGGGS (SEQ ID NO: 2). In certain embodiments, the linker comprises 3-5 repetitions of the GGGGS (SEQ ID NO: 2) motif. In certain embodiments, the amino acid linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 4), GGGGS GGGGS GGGGS GGGGS (SEQ ID NO: 5), or GGGGSGGGGSGGGGSGGGGAS (SEQ ID NO: 6). Alternatively, linker sequences connecting the variable domains may include glycine polymers (G)n; glycinealanine polymers; alanine-serine polymers; and other flexible linkers known in the art.
  • KESGSVSSEQLAQFRSLD (SEQ ID NO: 11) (Bird et al., Science 242:423- 426 (1988)), GGRRGGGS (SEQ ID NO: 12); LRQRDGERP (SEQ ID NO: 13); LRQKDGGGSERP (SEQ ID NO: 14); and GSTSGSGKPGSGEGSTKG (SEQ ID NO: 15) (Cooper et al, Blood, 101(4): 1637-1644 (2003)).
  • flexible linkers can be rationally designed using a computer program capable of modeling the 3D structure of proteins and peptides or by phage display methods.
  • the antibodies may comprise a variable light (VL) domain and a variable heavy (VH) domain.
  • VL and VH domain further comprises a set of three CDRs.
  • CDR refers to non-contiguous sequences of amino acids within antibody variable regions, which confer antigen specificity and affinity.
  • HCDR1, HCDR2, HCDR3 CDRs in each heavy chain variable domain
  • LCDR1, LCDR2, LCDR3 CDR1, LCDR2, LCDR3
  • FRs Framework regions
  • HFR1, HFR2, HFR3, and HFR4 FRs in each heavy chain variable domain
  • LFR1, LFR2, LFR3, and LFR4 four FRs in each light chain variable domain.
  • an antibody variable region amino acid sequence can be represented by the formula FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • Each segment of the formula i.e., FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4, represents a discrete amino acid sequence (or a polynucleotide sequence encoding the same) that can be mutated, including one or more amino acid substitutions, deletions, and insertions.
  • an antibody variable light chain amino acid sequence can be represented by the formula LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.
  • an antibody variable heavy chain amino acid sequence can be represented by the formula HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4.
  • the boundaries of a given CDR or FR may vary depending on the scheme used for identification.
  • the Kabat scheme is based on sequence alignments
  • the Chothia scheme is based on structural information. Numbering for both the Kabat and Chothia schemes is based upon the most common antibody region sequence lengths, with insertions accommodated by insertion letters, for example, “30a,” and deletions appearing in some antibodies. The two schemes place certain insertions and deletions (“indels”) at different positions, resulting in differential numbering.
  • the Contact scheme is based on analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme. [0207] Table 1, below, lists exemplary position boundaries of LCDR1, LCDR2,
  • LCDR3 and HCDR1, HCDR2, HCDR3 of an antibody as identified by Kabat, Chothia, and Contact schemes, respectively.
  • residue numbering is listed using both the Kabat and Chothia numbering schemes.
  • CDRs are located between FRs, for example, with CDRL1 located between LFR1 and LFR2, and so forth. It is noted that because the shown Kabat numbering scheme places insertions at H35A and H35B, the end of the Chothia CDRH1 loop when numbered using the shown Kabat numbering convention varies between H32 and H34, depending on the length of the loop.
  • CDR complementary determining region
  • individual specified CDRs e.g., CDRH1, CDRH2
  • CDRH1 as used herein is defined by residues H30-H35B (Kabat numbering) or H30-H35 (Chothia numbering).
  • an “FR” or “framework region,” or individual specified FRs (e.g., “HFR1,” “HFR2”) of a given antibody or fragment thereof, such as a variable domain thereof, should be understood to encompass a (or the specific) framework region as defined by Kabat. In some cases, the particular amino acid sequence of a CDR or FR is given.
  • the antigen binding proteins are humanized through resurfacing (i.e., remodel the solvent-accessible residues of the non-human framework such that they become more human-like).
  • resurfacing strategies are described in more detail in W02004/016740, WO2008/144757, and W02005/016950, each of which is incorporated herein by reference.
  • the antigen binding proteins are humanized through CDR grafting (i.e., inserting the rabbit antigen binding protein CDRs into a human antibody acceptor framework). Grafting strategies and human acceptor frameworks are described in more detail in W02009/155726, incorporated herein by reference.
  • sequence identity between two polypeptides is determined by comparing the amino acid sequence of one polypeptide to the sequence of a second polypeptide.
  • sequence identity between two polynucleotides is determined by comparing the nucleotide sequence of one polynucleotide to the sequence of a second polynucleotide.
  • the terms “% identical”, “% identity” or similar terms are intended to refer, in particular, to the percentage of nucleotides or amino acids (as applicable) which are identical after the sequences to be compared have been aligned to yield maximum identity, potentially introducing gaps.
  • Comparisons of two sequences are usually carried out by comparing the sequences, after optimal alignment, with respect to a segment or “window of comparison”, in order to identify local regions of corresponding sequences.
  • the optimal alignment for a comparison may be carried out manually or with the aid of the local homology algorithm by Smith and Waterman, 1981, Ads App. Math. 2, 482, with the aid of the local homology algorithm by Needleman and Wunsch, 1970, J. Mol. Biol. 48, 443, with the aid of the similarity search algorithm by Pearson and Lipman, 1988, Proc. Natl Acad. Sci.
  • NCBI National Center for Biotechnology Information
  • a variant polypeptide such as an antigen binding protein, may contain one or more substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence.
  • the variant polypeptide comprises one, two or three substitutions, insertions and/or deletions relative to the reference sequence. Substitutions, insertions, or deletions may result in the change of one or more biophysical parameters but particularly preferred are tolerated changes so that the polypeptide retains the desired activity.
  • the variant polypeptide comprises one or more tolerated substitutions, such as conservative substitutions.
  • such variant polypeptide maintains physically, biologically, chemically and/or functionally the properties of the corresponding reference sequence.
  • “Specifically recognizes” or “specifically binds” refers to the ability of the antigen binding proteins to bind selectively to the antigen in contrast to non-specific interactions with unrelated proteins which do not comprise the binding epitope. Suitable assays for determining the specific binding are described below.
  • the equilibrium dissociation constant (KD) of an antigen binding protein to an unrelated protein is less than about 50-fold lower than the one of the antigen binding protein to its antigen, as e.g., determined by SPR.
  • a specific antigenic determinant e.g., a target peptide-MHC
  • ELISA enzyme-linked immunosorbent assay
  • SPR surface plasmon resonance
  • BIAcore BIAcore instrument
  • kinetic rate constants can be determined at temperatures in the range of 15°C to 37°C. The present specification makes reference to kinetic rate constants determined by SPR throughout.
  • association rate constant values, dissociation rate constant values and equilibrium dissociation constant values recited herein are determined by SPR at 25°C SPR based on the monovalent antigen binding protein.
  • the SPR-based system used is a Biacore SPR system.
  • affinity values recited herein are determined in a Fab-scFv format, wherein the Fab targets CD3 and the scFv the pMHC.
  • the antigen binding protein is not a T cell receptor (TCR), including but not limited to, a soluble TCR.
  • TCR T cell receptor
  • TCRa and TCR0 the term “T cell receptor” or “TCR” refers to a heterodimeric protein comprised of two different chains (TCRa and TCR0), which structurally belong to the immunoglobulin (Ig) superfamily. The extracellular portion of each chain is composed of variable (“Va” and “V ”) and constant (“Ca” and “C0”) domains, and a hinge region, where the formation of a stabilizing disulfide bond occurs.
  • the intracellular region forms a non-covalent interaction with another trans-membrane protein, CD3, which in the case of the correct target recognition leads to a series of conformational changes and a first T cell activation signal.
  • Recognition and binding of peptide-MHC (pMHC) by a TCR is governed by the six hypervariable loops, termed complementarity determining regions (CDRs), located on the variable domains of the TCRa (CDRal, CDRa2, CDRa3) and TCRp (CDR 1, CDRP2, CDRP3).
  • CDRs complementarity determining regions
  • CDR3 loops (CDRa3 and CDRP3) lead the recognition of the processed antigen with the support of CDRal and CDRpi, that have been implicated in the recognition of the N- and C-terminal amino acids of the presented peptide, respectively (Rudolph et al. Annu Rev Immunol. 24:419-66. 2006). Recognition of the MHC is typically achieved through the interaction with CDRa2 and CDRP2.
  • the high sequence diversity of the TCR is achieved through V(D)J recombination process, in which the variable domain is generated from a combination of genes: V (variable) and J (joining) for both TCRa and TCRP, and an additional D (diversity) gene for TCRp.
  • the high antigen specificity of the TCR is controlled by the thymic maturation process, in which the self-reacting T cells are negatively selected.
  • TCR affinity towards the specific pMHC and the functional avidity are the key factors controlling T-cell activation.
  • a critical role in antigen recognition, however, is played by the affinity (KD), i.e., the strength of binding between the TCR and the cell-displayed pMHC (Tian et al. J Immunol. 179:2952-2960. 2007).
  • KD affinity
  • the physiological affinities of TCRs range from 1 pM to 100 pM (Davis et al. Annu Rev Immunol. 16:523-544. 1998), which, in comparison to antibodies, is relatively low.
  • peptide-MHC or “pMHC” or “pMHC complex” as used interchangeably herein refers to a major histocompatibility complex (MHC) molecule (MHC-I or -II) with an antigenic peptide bound in a peptide binding pocket of the MHC.
  • MHC molecules present peptides, in particular antigenic peptides, on the surface of cells to be recognized by immune cells.
  • pMHC refers to a complex of an MHC molecule and a peptide, in particular an antigenic peptide, presented by the MHC molecule.
  • HLA human leukocyte antigen
  • HLA is highly polygenic and can be broadly divided into three classes of MHC molecules, class I, class II and class III. Moreover, HLA genes have the highest level of polymorphism of the human genome.
  • the target peptide may be presented on an MHC class I complex (such as of serotype HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, HLA-K or HLA-L, or their respective subtypes) or an MHC class II complex (such as the serotypes HLA-DP, HLA-DQ, HLA-DR, DM or DO, or their respective subtypes).
  • MHC class I complex such as of serotype HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, HLA-K or HLA-L, or their respective subtypes
  • MHC class II complex such as the serotypes HLA-DP, HLA-D
  • the HLA-A protein constitutes the alpha chain of the respective class I MHC (major histocompatibility complex) protein, which further comprises a beta 2 microglobulin subunit.
  • the extracellular region of the alpha chain comprises three immunoglobulin-like domains (al, a2, and a3).
  • the al and a2 domains form a peptide- binding groove wherein the HLA- restricted peptide is typically located.
  • peptides displayed by the HLA complex may be identified as pHLA herein.
  • the terms “HLA-displayed”, “HLA-presented”, “loaded onto HLA”, “peptide-loaded HLA” and “HLA-restricted” are used interchangeably herein.
  • the target peptides of the present invention are displayed by the A01 supertype, see for example Table 1 of Sidney et al., BMC Immunology, Vol. 9, Article number: 1 (2008).
  • the target peptide is displayed by a serotype of the group consisting of HLA-A*01 :01, HLA-A*26:01, HLA- A*26:02, HLA-A*26:03, HLA-A*30:02, HLA-A*30:03, HLA-A*30:04, and HLA- A*32:01.
  • the invention provides antigen binding proteins that bind to MHC-displayed NTDNNLAVY (SEQ ID NO: 68), in particular antibody fragments such as scFvs and larger bispecific formats including such scFvs, e.g. Fab-scFvs2.
  • a pMHC is described by its HLA and target peptide, for example HLA-A*01:01/ KK-LC-1 is the peptide of NTDNNLAVY (SEQ ID NO: 68) displayed on HLA-A*01 :01.
  • HLA-A*01:01/PTS refers to the peptides of ETDNNIVVY (SEQ ID NO: 74), PTDENLARY (SEQ ID NO: 75), or NTDNLLTEY (SEQ ID NO: 76), respectively, being presented by an HLA*01 :01.
  • isolated antigen binding proteins that bind to MHC-displayed NTDNNLAVY (SEQ ID NO: 68), i.e., the isolated antigen binding protein is not associated or bound to the surface of a cell, such as a T cell. Isolated antigen binding proteins are separated from a component of its natural environment. In certain embodiments, the isolated antigen binding protein is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic methods (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC, affinity chromatography, size exclusion chromatography).
  • electrophoretic methods e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis
  • chromatography e.g., ion exchange or reverse phase HPLC, affinity chromatography, size exclusion chromatography.
  • the antigen binding protein is not a soluble TCR (e.g., a TCR lacking one or more of a transmembrane domain, an intracellular signaling domain, and constant domains).
  • the antigen binding protein is a monoclonal antibody, in particular an antibody fragment such as a scFv.
  • antigen binding proteins in particular isolated antibodies and artificial constructs comprising antibody fragments, that specifically recognize a target MHC-displayed NTDNNLAVY (SEQ ID NO: 68).
  • the antigen binding proteins possess surprisingly high affinity while retaining high specificity for the target (i.e., low to no affinity for pMHCs displaying other targets, including off-target peptides, or beta-2-microglobulin).
  • the beta-2 -microglobuin polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 24.
  • the MHC displaying the peptide is of the HLA A01 supertype.
  • the peptide is displayed by a serotype of the group consisting of HLA-A*01:01, HLA- A*26:01, HLA-A*26:02, HLA-A*26:03, HLA-A*30:02, HLA-A*30:03, HLA-A*30:04, and HLA-A*32:01, in particular HLA-A*01 :01.
  • the HLA- restricted off-target peptide is derived from the gene product of ICE1, DSG3, PTS, KDM7A, FUS, PTPRC, ROBO1, TBL3, CHTOP, DMXL2, FAP, GPAM, PDE10A, VAV1, ZBTB40 and/or ZNF430.
  • TBL3 derived peptide TBL3 derived peptide (TADHNLLLY; SEQ ID NO: 78) has been identified as having similarity to the KK-LC1 target peptide NTDNNLAVY (SEQ ID NO: 68) but being highly expressed among many healthy tissues and immune cells.
  • the HLA- restricted PTPRC derived peptide consists of NLDKNLIKY (SEQ ID NO: 80).
  • the HLA- restricted ROBO1 derived peptide consists of NSDSNLTTY (SEQ ID NO: 77).
  • the HLA- restricted TBL3 derived peptide consists of TADHNLLLY (SEQ ID NO: 78).
  • the HLA- restricted CHTOP derived peptide consists of QLDNQLDAY (SEQ ID NO: 81).
  • the HLA- restricted DMXL2 derived peptide consists of SVDSNLFVY (SEQ ID NO: 82).
  • the HLA- restricted FAP derived peptide consists of SADNNIVLY (SEQ ID NO: 83).
  • the HLA- restricted GPAM derived peptide consists of RTERNVAVY (SEQ ID NO: 84).
  • the HLA- restricted PDE10A derived peptide consists of GGDNQLLLY (SEQ ID NO: 85).
  • the HLA- restricted VAV1 derived peptide consists of NLDQSLAHY (SEQ ID NO: 86).
  • the HLA- restricted ZBTB40 derived peptide consists of FTDNQILLK (SEQ ID NO: 87).
  • the HLA- restricted ZNF430 derived peptide consists of GADRNLLVY (SEQ ID NO: 88).
  • the antigen binding protein has at least about a 10-fold reduction in affinity (KD) to one or more of MHC -di splayed peptides selected from the group consisting of YTDNWLAVY (SEQ ID NO: 73), ETDNNIVVY (SEQ ID NO: 74), PTDENLARY (SEQ ID NO: 75), NTDNLLTEY (SEQ ID NO: 76), NSDSNLTTY (SEQ ID NO: 77), TADHNLLLY (SEQ ID NO: 78), NSDNNTIFV (SEQ ID NO: 79), NLDKNLIKY (SEQ ID NO: 80), QLDNQLDAY (SEQ ID NO: 81), SVDSNLFVY (SEQ ID NO: 82), SADNNIVLY (SEQ ID NO: 83), GGDNQLLLY (SEQ ID NO: 85), NLDQSLAHY (SEQ ID NO: 86), FTDNQILLK (KD)
  • the antigen binding protein has at least a 50-fold decrease in affinity (KD) (e.g., a 75-fold, 80-fold, 90-fold, 100-fold, 120-fold, 150-fold, or 200-fold decrease in affinity (KD)) for one or more MHC-displayed peptides selected from the group consisting of YTDNWLAVY (SEQ ID NO: 73), ETDNNIVVY (SEQ ID NO: 74), NTDNLLTEY (SEQ ID NO: 76), NSDSNLTTY (SEQ ID NO: 77), TADHNLLLY (SEQ ID NO: 78), NSDNNTIFV (SEQ ID NO: 79), NLDKNLIKY (SEQ ID NO: 80), QLDNQLDAY (SEQ ID NO: 81), SVDSNLFVY (SEQ ID NO: 82), SADNNIVLY (SEQ ID NO: 83), GGDNQLLLY (SEQ ID NO: 73), ETDNNIV
  • the antigen binding protein has at least a 50-fold decrease in affinity (KD) (e.g., a 75-fold, 80-fold, 90-fold, 100-fold, 120-fold, 150-fold, or 200-fold decrease in affinity (KD)) for the MHC-displayed peptide TADHNLLLY (SEQ ID NO: 78), relative to MHC-displayed NTDNNLAVY (SEQ ID NO: 68), as determined by SPR.
  • KD affinity
  • TADHNLLLY SEQ ID NO: 78
  • MHC-displayed NTDNNLAVY SEQ ID NO: 68
  • the disclosure provides a method of producing an antigen binding protein with affinity to a target MHC-displayed antigen, comprising: i) contacting a plurality of antigen binding proteins with the target MHC-displayed antigen complex; ii) contacting the plurality of antigen binding proteins with MHC-displayed peptide TADHNLLLY (SEQ ID NO: 78) complex; iii) determining the level of binding of the antigen binding proteins to the target MHC-displayed antigen and to the MHC-displayed TADHNLLLY (SEQ ID NO: 78) complex; iv) selecting an antigen binding protein from the plurality of antigen binding proteins that bind the target MHC-displayed antigen complex and lack substantial binding to the MHC-displayed TADHNLLLY (SEQ ID NO: 78) complex; and v) producing the selected antigen binding protein.
  • the target MHC-displayed antigen is MHC- displayed NTDNNLAVY (SEQ ID NO: 68).
  • the MHC molecule is a class I MHC molecule. In certain embodiments, the MHC molecule is a HLA-A*0 molecule. In certain embodiments, the MHC molecule is HLA-A*01:01.
  • the one or more selected antigen binding proteins do not detectably bind MHC-displayed TADHNLLLY (SEQ ID NO: 78) complex.
  • the one or more selected antigen binding proteins have at least about a 10-fold reduction in affinity to the MHC-displayed TADHNLLLY (SEQ ID NO: 78), relative to the affinity to the target MHC-displayed antigen, in particular at least a 50-fold decrease in affinity (e.g., a 75-fold, 80-fold, 90-fold, 100-fold, 120-fold, 150-fold, or 200-fold decrease in affinity).
  • the lack of substantial binding corresponds to an affinity (KD) of about 400 nM or higher, as determined by SPR.
  • the lack of substantial binding corresponds to an affinity (KD) of about 400 nM to about 1 pM, as determined by SPR.
  • the method further comprises the step of repeating steps (i)-(iv) using the selected antigen binding protein.
  • the plurality of antigen binding proteins are displayed on the surface of a cell.
  • the cell is a mammalian cell, a yeast cell, an insect cell, or a bacterial cell.
  • determining binding of the plurality of antigen binding proteins to the target MHC-displayed antigen comprises contacting the plurality of antigen binding proteins with an isolated target MHC-displayed antigen and detecting binding via ELISA or SPR.
  • step ii) further comprises contacting the plurality of antigen binding proteins with one or more MHC-displayed off-target peptide complexes, wherein said one or more off-target peptides are selected from the group consisting of YTDNWLAVY (SEQ ID NO: 73), ETDNNIVVY (SEQ ID NO: 74), NTDNLLTEY (SEQ ID NO: 76), NSDSNLTTY (SEQ ID NO: 77), TADHNLLLY (SEQ ID NO: 78), NSDNNTIFV (SEQ ID NO: 79), NLDKNLIKY (SEQ ID NO: 80), QLDNQLDAY (SEQ ID NO: 81), SVDSNLFVY (SEQ ID NO: 82), SADNNIVLY (SEQ ID NO: 83), GGDNQLLLY (SEQ ID NO: 85), NLDQSLAHY (SEQ ID NO: 86), FTDNQILLK (SEQ ID NO: 73), ETD
  • step iii) further comprises determining the level of binding of the antigen binding proteins to the target MHC-displayed antigen and to the one or more MHC-displayed off-target peptide complexes.
  • step iv) further comprises selecting an antigen binding protein from the plurality of antigen binding proteins that bind the target MHC- displayed antigen complex and lack substantial binding to the one or more MHC-displayed off-target peptide complexes.
  • the disclosure provides an antigen binding protein produced by the method of producing described herein.
  • the disclosure provides an antigen binding protein which specifically binds to a major histocompatibility complex (MHC)-displayed NTDNNLAVY (SEQ ID NO: 68), wherein the antigen binding protein has a binding specificity dictated by at least 4 amino acid positions in said MHC-displayed NTDNNLAVY (SEQ ID NO: 68), such that the EC50-value of Granzyme B (GrzB) expression is increased by at least a 10-fold when one amino acid residue of said at least 4 amino acid positions is substituted, e.g., by alanine, serine, arginine and/or aspartic acid.
  • MHC major histocompatibility complex
  • NTDNNLAVY SEQ ID NO: 68
  • the amino acid at position 8 is substituted by alanine, arginine and/or aspartic acid.
  • the target peptide NTDNNLAVY (SEQ ID NO: 68) comprises a substitution selected from the group consisting of N5A, L6A, A7S, V8A, N5R, L6R, A7R, V8R, N5D, L6D, A7D or V8D.
  • Anchor positions for HLA-A*01 :01 molecules typically include P2 (frequently), P3 (frequently) and PQ (always); anchor residues for HLA- A*01 :01 molecules typically include a tyrosine (Y) at position 9, and either a small polar or hydrophobic (S, T, M, L) residue in position 2, or a negatively charged (D or E) residue in position 3 (see Kondo et al, Immunogenetics, 1997;45(4):249-58).
  • a person skilled in the art would be able to modify the amino acid sequences of the target peptide of NTDNNLAVY (SEQ ID NO: 68), by maintaining the known anchor residues, and determine whether such variants maintain the ability to bind the MHC.
  • said binding specificity is dictated by 6 amino acid residues.
  • said 6 amino residues are at position 4, 5, 6, 7, 8 and/or 9.
  • the specificity motif of 6 amino acids includes anchor residues.
  • the disclosure provides an antigen binding protein which specifically binds to a major histocompatibility complex (MHC)-displayed NTDNNLAVY (SEQ ID NO: 68), wherein the antigen binding protein has at least about a 10-fold reduction in affinity (KD) to MHC-displayed NTDNALAVY (SEQ ID NO: 69), NTDNNAAVY (SEQ ID NO: 70), NTDNNLSVY (SEQ ID NO: 71), and NTDNNLAAY (SEQ ID NO: 72), relative to the affinity (K D ) to MHC-displayed NTDNNLAVY (SEQ ID NO: 68), as e.g. determined by SPR.
  • the antigen binding protein has at least about a 10-fold reduction in affinity (KD) to MHC-displayed NTDNALAVY (SEQ ID NO: 69), NTDNNAAVY (SEQ ID NO: 70), NTDNNLSVY (SEQ ID NO: 71), and NTDNNLAAY
  • the antigen binding protein selectively binds a pMHC complex of a given HLA subtype and a target peptide, but not to a pMHC complex of the same HLA subtype presenting a different peptide, in particular off-target peptide.
  • the antigen binding protein specifically binds the NTDNNLAVY (SEQ ID NO: 68) displayed by on HLA-A*01 on the surface of a cell, such as a cancer cell.
  • the cell is a T2 cell expressing HLA-A*01 that has been pulsed with the target peptide NTDNNLAVY (SEQ ID NO: 68).
  • the cell is a Cos-7 cell expressing HLA-A*01 that has been pulsed with the target peptide NTDNNLAVY (SEQ ID NO: 68).
  • the cell is a cancer cell that endogenously presents the target peptide NTDNNLAVY (SEQ ID NO: 68) by an HLA complex (i.e., without pulsing).
  • the antigen binding protein has an affinity (KD) to an MHC-displayed NTDNNLAVY (SEQ ID NO: 68) of at least about 30 nM or stronger (e.g., about 29 nM, about 28 nM, about 27 nM, about 26 nM, about 25 nM, about 24 nM, about 23 nM, about 22 nM, about 21 nM, about 20 nM, about 19 nM, about 18 nM, about 17 nM, about 16 nM, about 15 nM, about 14 M, about 13 nM, about 12 nM, about 11 nM, about 10 nM, about 9 nM, about 8 nM, about 7 nM, about 6 nM or about 5 nM, about 4 nM, about 3 nM, about 2 nM or stronger, e.g.
  • the antigen binding protein comprises: (i) an antibody heavy chain variable (VH) domain comprising an HCDR1 amino acid sequence of XiSYYYMC, wherein Xi is R or S (SEQ ID NO: 140), an HCDR2 amino acid sequence of QYAGSSGSTYYASWAKG (SEQ ID NO: 141), and an HCDR3 amino acid sequence of GAGYGNX2GHSL, wherein X2 is D or G (SEQ ID NO: 142); and/or (ii) an antibody light chain variable (VL) domain comprising an LCDR1 amino acid sequence of X3ASX4NIYNSLA, wherein X3 is Q or R, and XAs E or K (SEQ ID NO: 143), an LCDR2 amino acid sequence of GASNLES (SEQ ID NO: 144), and an LCDR3 amino acid sequence of QX TYYGHDNX. GGA. wherein X5 is S or A, and
  • the antigen binding protein comprises: an antibody heavy chain variable (VH) domain comprising an HCDR1 amino acid sequence of SSYYYMC (SEQ ID NO: 155), an HCDR2 amino acid sequence of CIYAGSSGSTYYASWAKG (SEQ ID NO: 156), and an HCDR3 amino acid sequence of GAGYGNGGHSL (SEQ ID NO: 157); and an antibody light chain variable (VL) domain comprising an LCDR1 amino acid sequence of RASKNIYNSLA (SEQ ID NO: 152), an LCDR2 amino acid sequence of GASNLES (SEQ ID NO: 153), and an LCDR3 amino acid sequence of QATYYGHDNIGGA (SEQ ID NO: 154).
  • VH antibody heavy chain variable
  • said antigen binding protein comprises: (1) an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 160, and/or an antibody light chain variable (VL) domain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 161; or (2) an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 164, and/or and an antibody light chain variable (VL) domain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 165.
  • VH antibody heavy chain variable
  • VL antibody light chain variable domain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO
  • the antigen binding protein comprises: (a) an antibody heavy chain variable (VH) domain comprising an HCDR1 amino acid sequence of SSYYYMC (SEQ ID NO: 25) an HCDR2 amino acid sequence of CIYAGSSGSTYYASWAKG (SEQ ID NO: 26), and an HCDR3 amino acid sequence of GAGYGNDGHSL (SEQ ID NO: 27); and (b) an antibody light chain variable (VL) domain comprising an LCDR1 amino acid sequence of QASENIYNSLA (SEQ ID NO: 28), an LCDR2 amino acid sequence of GASNLES (SEQ ID NO: 29), and an LCDR3 amino acid sequence of QSTYYGHDNVGGA (SEQ ID NO: 30).
  • VH antibody heavy chain variable
  • said antigen binding protein comprises: (1) an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 31, and/or an antibody light chain variable (VL) domain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 32; or (2) an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 33, and/or and an antibody light chain variable (VL) domain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 34; or (3) an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 35, and/
  • the antigen binding protein comprises: an antibody heavy chain variable (VH) domain comprising an HCDR1 amino acid sequence of RSYYYMC (SEQ ID NO: 149), an HCDR2 amino acid sequence of CIYAGSSGSTYYASWAKG (SEQ ID NO: 150), and an HCDR3 amino acid sequence of GAGYGNDGHSL (SEQ ID NO: 151); and an antibody light chain variable (VL) domain comprising an LCDR1 amino acid sequence of RASKNIYNSLA (SEQ ID NO: 146), an LCDR2 amino acid sequence of GASNLES (SEQ ID NO: 147), and an LCDR3 amino acid sequence of QATYYGHDNVGGA (SEQ ID NO: 148).
  • VH antibody heavy chain variable
  • VL antibody light chain variable domain comprising an LCDR1 amino acid sequence of RASKNIYNSLA (SEQ ID NO: 146), an LCDR2 amino acid sequence of GASNLES (SEQ ID NO: 147), and an LCDR3 amino acid sequence of QA
  • said antigen binding protein comprises: an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 162, and/or an antibody light chain variable (VL) domain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 163.
  • VH antibody heavy chain variable
  • VL antibody light chain variable
  • the antigen binding protein is or comprises a full- length immunoglobulin or an antibody fragment such as a Fab, a Fab', a F(ab’)2, a scFv, a Fv fragment.
  • the antigen binding protein comprises a scFv with an amino acid sequence that is at least about 79%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 167, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 166, SEQ ID NO: 168 or SEQ ID NO: 169.
  • said scFv has a human germline identity of at least about 80%. For determination of the identity score, see e.g. Example 13,
  • the disclosure provides a bispecific antigen binding protein, comprising a first antigen binding domain comprising the antigen binding protein described above, and at last one antigen binding domain with specificity for a cell surface protein of an immune cell (e.g., CD3 on the surface of a T cell; an “immune cell binding domain”).
  • a cell surface protein of an immune cell e.g., CD3 on the surface of a T cell; an “immune cell binding domain”.
  • the immune cell is a human cell.
  • the immune cell is selected from the group consisting of a T cell, a B cell, a natural killer (NK) cell, a natural killer T (NKT) cell, a neutrophil cell, a monocyte, and a macrophage.
  • the immune cell is a T cell.
  • the immune cell binding domain is an antibody, such as a CD3 targeting antibody, a CD 16a targeting antibody or BMA031 or a variant thereof.
  • CD3 refers to any native CD3 from any vertebrate source, including primates.
  • the immune cell binding domain specifically binds to human CD3, in particular to the CD3s (epsilon) chain/subunit of CD3 (see e.g., UniProt (www.uniprot.org) accession no. P07766 (version 189), or NCBI (www.ncbi.nlm.nih.gov/) RefSeq NP_000724.1.)
  • the CD3 molecule may be the full- length, unprocessed CD3 molecule or a fragment or variant thereof that e.g., results from processing in the cell.
  • Suitable anti-CD3 binding domains are known in the art, particularly T-cell activating CD3-epsilon binding domains.
  • the terms “CD3 binding domain” and “anti- CD3 binding domain” are used interchangeably herein.
  • the anti-CD3 binding domain is any one of antibodies SP34, OKT3 or UCHT1, or a variant sequence thereof having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, while retaining the same specificity as its parent.
  • the antigen binding protein of the disclosure comprises a Fab domain as well as a first and a second pMHC binding domain, both pMHC binding domains being scFvs, i.e., the molecule is a Fab-(scFv)2.
  • An advantage of the Fab-scFv? scaffolds of the disclosure is the intermediate molecular size of approximately 75-110 kDa.
  • Blinatumomab a bispecific T cell engager (BiTE) has shown excellent results in patients with relapsed or refractory acute lymphoblastic leukemia.
  • the immune cell binding domain in particular the Fab domain, specifically binds to CD3 with an affinity (KD) between about 1 nM to about 150 nM (e.g., 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, 11 nM, 12 nM, 13 nM, 14 nM, 15 nM, 16 nM, 17 nM, 18 nM, 19 nM, 20 nM, 21 nM, 22 nM, 23 nM, 24 nM, 25 nM, 26 nM, 27 nM, 28 nM, 29 nM, 30 nM, 31 nM, 32 nM, 33 nM, 34 nM, 35 nM, 36 nM, 37 nM, 38 nM, 39 nM, 40 nM, 41
  • KD affinity
  • the immune cell binding domain in particular the Fab domain, specifically binds to CD3 with an affinity (KD) between about 1 nM to about 50 nM, as determined by SPR. In certain embodiments, the immune cell binding domain, in particular the Fab domain, specifically binds to CD3 with an affinity (KD) between about 1 nM to about 10 nM, e.g., about 3 nM, as determined by SPR.
  • the immune cell binding domain in particular the Fab domain, specifically binds to CD3 with an affinity (KD) of about 1 nM, of about 10 nM, or of about 50 nM, as determined by SPR.
  • KD affinity
  • the association rate constant k a of the anti-CD3 binding domain is between about l * 10 5 to about l * 10 7 M ⁇ s’ 1 , such as at least l * 10 6 M’ 1 or at least 2* 10 6 M ⁇ s’ 1 .
  • association rate constant k a and/or the dissociation rate constant ka values that are within 1-fold of each other, 1.5-fold of each other, 2-fold of each other, 2.5-fold of each other or 3-fold of each other, i.e., association rate constant k a values of l * 10 5 M ⁇ s’ 1 and 3* 10 5 M’ 1 .
  • the antigen binding protein is a Fab-(scFv)2 and comprises (i) a single Fab domain which specifically binds to CD3 with an affinity (KD) from about 1 nM to about 50 nM, such as about 3 nM, (ii) a first pMHC binding scFv linked to the C-terminus of the Fab domain heavy chain and (iii) a second pMHC binding scFv linked to the C-terminus of the Fab domain light chain, wherein both pMHC binding scFvs have an affinity (KD) of about 30 nM to about 10 nM to the target pMHC complex.
  • KD affinity
  • an antigen binding protein as provided by the present disclosure in particular the at least first and/or the at least second pMHC binding domain, is highly selective and does not bind to a different pMHC complex presenting an unrelated non-target peptide.
  • such bispecific Fab-(scFv)2 comprises a HC comprising or consisting of an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 172; and a LC comprising or consisting of an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 173.
  • such bispecific Fab-(scFv)2 comprises a HC comprising or consisting of an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 175; and a LC comprising or consisting of an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 177.
  • VH antibody heavy chain variable domain comprising an HCDR1 amino acid sequence of STYAMN (SEQ ID NO: 46) an HCDR2 amino acid sequence of RIRSKYNNYATYYADSVKG (SEQ ID NO: 47), and an HCDR3 amino acid sequence of HGNFGDSYVSWFAY (SEQ ID NO: 48); and
  • VL antibody light chain variable domain
  • LCDR1 amino acid sequence of GSSTGAVTTSNYAN SEQ ID NO: 49
  • LCDR2 amino acid sequence of GTNKRAP SEQ ID NO: 50
  • LCDR3 amino acid sequence of ALWYSNHWV SEQ ID NO: 51
  • antigen binding protein of claim [1] or [2], wherein said immune cell binding domain comprises:
  • VH antibody heavy chain variable domain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 43, and/or
  • VL antibody light chain variable domain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 45.
  • VH antibody heavy chain variable domain
  • HCDR1 amino acid sequence of SSYYYMC SEQ ID NO: 155
  • HCDR2 amino acid sequence of CIYAGSSGSTYYASWAKG SEQ ID NO: 156
  • HCDR3 amino acid sequence of GAGYGNGGHSL SEQ ID NO: 157
  • an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 160
  • an antibody light chain variable (VL) domain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 161; or
  • an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 164
  • an antibody light chain variable (VL) domain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 165.
  • VH antibody heavy chain variable domain comprising an HCDR1 amino acid sequence of SSYYYMC (SEQ ID NO: 25), an HCDR2 amino acid sequence of CIYAGSSGSTYYASWAKG (SEQ ID NO: 26), and an HCDR3 amino acid sequence of GAGYGNDGHSL (SEQ ID NO: 27); and
  • an antibody light chain variable (VL) domain comprising an LCDR1 amino acid sequence of QASENIYNSLA (SEQ ID NO: 28), an LCDR2 amino acid sequence of GASNLES (SEQ ID NO: 29), and an LCDR3 amino acid sequence of QSTYYGHDNVGGA (SEQ ID NO: 30).
  • an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 31, and/or an antibody light chain variable (VL) domain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 32; or
  • an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 35
  • an antibody light chain variable (VL) domain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 36
  • the antigen binding protein of any one of the above [1] to [3], wherein said first and optionally second antigen binding domain that binds to the MHC-displayed NTDNNLAVY (SEQ ID NO: 68), comprises:
  • VH antibody heavy chain variable domain comprising an HCDR1 amino acid sequence of RSYYYMC (SEQ ID NO: 149), an HCDR2 amino acid sequence of CIYAGSSGSTYYASWAKG (SEQ ID NO: 150), and an HCDR3 amino acid sequence of GAGYGNDGHSL (SEQ ID NO: 151); and
  • VL antibody light chain variable domain comprising an LCDR1 amino acid sequence of RASKNIYNSLA (SEQ ID NO: 146), an LCDR2 amino acid sequence of GASNLES (SEQ ID NO: 147), and an LCDR3 amino acid sequence of QATYYGHDNVGGA (SEQ ID NO: 148).
  • VH antibody heavy chain variable
  • VL antibody light chain variable
  • a HC comprising or consisting of an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98% or 100% identical to the amino acid sequence of SEQ ID NO: 62; and a LC comprising or consisting of an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98% or 100% identical to the amino acid sequence of SEQ ID NO: 63;
  • a HC comprising or consisting of an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98% or 100% identical to the amino acid sequence of SEQ ID NO: 64; and a LC comprising or consisting of an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98% or 100% identical to the amino acid sequence of SEQ ID NO: 65; or
  • the antigen binding protein of [14] being or comprising a (scFv)s, tribody, Fab?, Fabs, Fab4, or scFv-Fab-scFv (Fab-scFv?).
  • a HC comprising or consisting of an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 175; and a LC comprising or consisting of an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98% or 100% identical to the amino acid sequence of SEQ ID NO: 177; or
  • a HC comprising or consisting of an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 178; and a LC comprising or consisting of an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98% or 100% identical to the amino acid sequence of SEQ ID NO: 179.
  • An antigen binding protein that specifically binds to a major histocompatibility complex (MHC)-displayed NTDNNLAVY (SEQ ID NO: 68), comprising: an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 160, and/or an antibody light chain variable (VL) domain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 161.
  • MHC major histocompatibility complex
  • VL antibody light chain variable
  • An antigen binding protein that specifically binds to a major histocompatibility complex (MHC)-displayed NTDNNLAVY (SEQ ID NO: 68), comprising: an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 164, and/or an antibody light chain variable (VL) domain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 165.
  • MHC major histocompatibility complex
  • VL antibody light chain variable
  • the antigen binding protein of [17] and/or [18], comprising (a) an antibody heavy chain variable (VH) domain comprising an HCDR1 amino acid sequence of SSYYYMC (SEQ ID NO: 155), an HCDR2 amino acid sequence of CIYAGSSGSTYYASWAKG (SEQ ID NO: 156), and an HCDR3 amino acid sequence of GAGYGNGGHSL (SEQ ID NO: 157); and (b) an antibody light chain variable (VL) domain comprising an LCDR1 amino acid sequence of RASKNIYNSLA (SEQ ID NO: 152), an LCDR2 amino acid sequence of GASNLES (SEQ ID NO: 153), and an LCDR3 amino acid sequence of QATYYGHDNIGGA (SEQ ID NO: 154).
  • VH antibody heavy chain variable domain
  • HCDR1 amino acid sequence of SSYYYMC SEQ ID NO: 155
  • an HCDR2 amino acid sequence of CIYAGSSGSTYYASWAKG SEQ ID
  • An antigen binding protein that specifically binds to a major histocompatibility complex (MHC)-displayed NTDNNLAVY (SEQ ID NO: 68), comprising: an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 31, and/or an antibody light chain variable (VL) domain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 32.
  • MHC major histocompatibility complex
  • VL antibody light chain variable
  • An antigen binding protein that specifically binds to a major histocompatibility complex (MHC)-displayed NTDNNLAVY (SEQ ID NO: 68), comprising: an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 33, and/or and an antibody light chain variable (VL) domain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 34.
  • MHC major histocompatibility complex
  • VL antibody light chain variable
  • An antigen binding protein that specifically binds to a major histocompatibility complex (MHC)-displayed NTDNNLAVY (SEQ ID NO: 68), comprising: an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 35, and/or an antibody light chain variable (VL) domain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 36.
  • MHC major histocompatibility complex
  • VL antibody light chain variable
  • the antigen binding protein of [20], [21] and/or [22], comprising (a) an antibody heavy chain variable (VH) domain comprising an HCDR1 amino acid sequence of SSYYYMC (SEQ ID NO: 25), an HCDR2 amino acid sequence of CIYAGSSGSTYYASWAKG (SEQ ID NO: 26), and an HCDR3 amino acid sequence of GAGYGNDGHSL (SEQ ID NO: 27); and (b) an antibody light chain variable (VL) domain comprising an LCDR1 amino acid sequence of QASENIYNSLA (SEQ ID NO: 28), an LCDR2 amino acid sequence of GASNLES (SEQ ID NO: 29), and an LCDR3 amino acid sequence of QSTYYGHDNVGGA (SEQ ID NO: 30).
  • VH antibody heavy chain variable domain
  • VL antibody light chain variable domain comprising an LCDR1 amino acid sequence of QASENIYNSLA (SEQ ID NO: 28), an LCDR2 amino acid sequence of GASNLES (SEQ ID NO: 29),
  • the antigen binding protein of [24], comprising (a) an antibody heavy chain variable (VH) domain comprising an HCDR1 amino acid sequence of RSYYYMC (SEQ ID NO: 149), an HCDR2 amino acid sequence of CIYAGSSGSTYYASWAKG (SEQ ID NO: 150), and an HCDR3 amino acid sequence of GAGYGNDGHSL (SEQ ID NO: 151); and (b) an antibody light chain variable (VL) domain comprising an LCDR1 amino acid sequence of RASKNIYNSLA (SEQ ID NO: 146), an LCDR2 amino acid sequence of GASNLES (SEQ ID NO: 147), and an LCDR3 amino acid sequence of Q ATYYGHDNVGGA (SEQ ID NO: 148).
  • VH antibody heavy chain variable domain
  • VH antibody heavy chain variable domain comprising an HCDR1 amino acid sequence of RSYYYMC (SEQ ID NO: 149), an HCDR2 amino acid sequence of CIYAGSSGSTYYASWAKG
  • variants of the sequences disclosed herein differs from its parental sequence by virtue of insertion (including addition), deletion and/or substitution of one or more amino acid residues or nucleobases, respectively, while retaining at least one desired property of the parent sequence disclosed herein, e.g., specific antigen binding, efficacy on target positive tumor cells, stability (e.g., serum stability, thermal stability, and/or storage stability), producibility (e.g., expression levels), safety (e.g., reactivity in healthy tissues, Granzyme B release, proinflammatory cytokine IFN gamma antigen-positive or antigen-negative cell lines, and/or low to no induction of pro-inflammatory cytokines IL-2, IL-6 and TNF alpha cytokine release), efficacy (e.g., tumor growth inhibition, tumor eradication, and/or T cell activating properties as e.g.
  • stability e.g., serum stability, thermal stability, and/or storage stability
  • producibility e.g., expression levels
  • the variant antigen binding protein retains binding to the target pMHC complex of at least 50%, such as 60%, 70%, 80%, 90% or 95% of the equilibrium dissociation constant KD of the reference antigen binding protein (i.e., the corresponding antigen binding protein without said substitutions, insertions, and/or deletions) when measured under identical conditions.
  • the variant shows stronger binding to the target peptide than the reference antigen binding protein.
  • Variants may be artificially engineered or naturally occurring, such as e.g., allelic or splice variants.
  • the variant antigen binding protein comprises an amino acid sequence being at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence disclosed herein.
  • the variant of the antigen binding protein detailed supra e.g. said VH and or VL domains and therewith antigen binding proteins comprising said domains as e.g., scFvs, Fab-scfvs or Fab-scFv2, comprises substitutions, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or more substitutions, within the VH and/or VL domain.
  • substitutions are within the CDRs.
  • substitutions are within the framework regions.
  • a variant of the VL domain comprises the LCDR1 amino acid sequence of RASKNIYNSLA (SEQ ID NO: 152), an LCDR2 amino acid sequence of GASNLES (SEQ ID NO: 153), and an LCDR3 amino acid sequence of QATYYGHDNIGGA (SEQ ID NO: 154).
  • a variant of the VL domain comprises the LCDR1 amino acid sequence of QASENIYNSLA (SEQ ID NO: 28), the LCDR2 amino acid sequence of GASNLES (SEQ ID NO: 29), and the LCDR3 amino acid sequence of QSTYYGHDNVGGA (SEQ ID NO: 30).
  • a variant of the VL domain comprises the LCDR1 amino acid sequence of RASKNIYNSLA (SEQ ID NO: 146), an LCDR2 amino acid sequence of GASNLES (SEQ ID NO: 147), and an LCDR3 amino acid sequence of QATYYGHDNVGGA (SEQ ID NO: 148).
  • a variant of the VH domain comprises the antibody heavy chain variable (VH) domain comprising an HCDR1 amino acid sequence of SSYYYMC (SEQ ID NO: 155), an HCDR2 amino acid sequence of CIYAGSSGSTYYASWAKG (SEQ ID NO: 156), and an HCDR3 amino acid sequence of GAGYGNGGHSL (SEQ ID NO: 157).
  • a variant of the VH domain comprises the HCDR1 amino acid sequence of SSYYYMC (SEQ ID NO: 25) an HCDR2 amino acid sequence of CIYAGSSGSTYYASWAKG (SEQ ID NO: 26), and an HCDR3 amino acid sequence of GAGYGNDGHSL (SEQ ID NO: 27).
  • a variant of the VH domain comprises an antibody heavy chain variable (VH) domain comprising an HCDR1 amino acid sequence of RSYYYMC (SEQ ID NO:
  • co-stimulatory domains were added in the second-generation CARs; and third generation CARs include two or more co-stimulatory domains (Maus MV et al (2014) Blood, 123: 2625- 2635). Said co-stimulatory domains may be selected from the group consisting of CD28, 0X40 and/or 4-1BB. Apart from CD3-zeta, other ITAM- containing domains have been explored including the Fc receptor for IgE-y domain.
  • Suitable immune cells include, without being limited to, T cells, Natural
  • the disclosure provides a chimeric antigen receptor (CAR) that specifically recognizes a peptide-MHC, comprising: i) an antigen binding protein with specificity to the peptide-MHC; ii) a transmembrane domain; and iii) an intracellular signaling domain, wherein the peptide-MHC is MHC-displayed NTDNNLAVY (SEQ ID NO: 68).
  • the CAR comprises a scFv being at least 90%
  • the transmembrane domain is selected from the group consisting of an artificial hydrophobic sequence and transmembrane domains of a type I transmembrane protein, an alpha, beta or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.
  • the intracellular signaling domain is selected from the group consisting of cytoplasmic signaling domains of a human CD3 zeta chain, FcyRIII, a cytoplasmic tail of an Fc receptor, an immunoreceptor tyrosine-based activation motif (ITAM) bearing cytoplasmic receptors, TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d.
  • ITAM immunoreceptor tyrosine-based activation motif
  • the antibody domain may be any of the antigen binding proteins outlined above.
  • the antibody domain comprises an antibody variable light domain (VL) comprising an amino acid sequence represented by the formula LFR1- CDRL1-LFR2-CDRL2-LFR3-CDRL3-LFR4.
  • the antibody domain comprises an antibody variable heavy domain (VH) comprising an amino acid sequence represented by the formula HFR1-CDRH1-HFR2-CDRH2-HFR3-CDRH3- HFR4.
  • the antibody domain comprises a scFv as described elsewhere herein.
  • the antigen binding protein retains stability and remains at least 94%, 95%, 96%, 97%, 98%, 99% or 100% monomeric after incubation for 14 days at 4° C in PBS at 1 mg/ml and/or 10 mg/ml as determined by SEC-HPLC.
  • Anti-drug antibodies may affect the risk profile and efficacy of a biological drug. If neutralizing, they may block the drug’s ability to bind to its target. It is therefore a regulatory requirement to test biologic drugs for the binding of anti-drug antibodies and their neutralizing potential.
  • Anti-drug antibody assays are e.g., detailed in W02007101661A1 (Hoffmann La Roche), WO2018178307A1 (Ablynx), WO202 1046316 A2 (Adverum Biotechnologies, Charles River), and US20180088140A1 (Genzyme Corporation), each of which is incorporated herein by reference.
  • Anti-drug antibodies binding to a tumor targeting domain of an antigen binding protein may lead to clustering of said antigen binding protein when each variable domain of the ADA binds to one tumor targeting domain of two antigen binding proteins.
  • the two or more CD3 binding domains on said antigen binding protein cluster and overstimulate the targeted T cell in the absence of target engagement, thereby leading to off-target toxicity. Unspecific stimulation of the T-cells may lead to systemic cytokine release.
  • the inventors have found that certain mutations in the tumor antigen binding domain of a T cell engager reduce ADA response and at the same time reduce nonspecific T cell stimulation in the absence of target engagement. Thereby, a highly effective and safe approach for cancer immunotherapy is provided.
  • variable heavy chain amino acid at position 11, 89, and/or 108, according to Kabat numbering is substituted with a polar amino acid; and/or serine (S) at position 113 is deleted, according to Kabat numbering.
  • S serine
  • substitution is particularly favorable when the binding domain is in scFv format.
  • one or both scFvs may comprise such substitution or deletion.
  • the polar amino acid is serine (S) and/or threonine (T).
  • the heavy chain amino acid is substituted with serine (S) at heavy chain amino acid position 11, serine (S) or threonine (T) at heavy chain amino acid position 89, and/or serine (S) or threonine (T) at heavy chain amino acid position 108, according to Kabat numbering.
  • the heavy chain amino acid is substituted with serine (S) at heavy chain amino acid position 11, serine (S) at heavy chain amino acid position 89, and serine (S) at heavy chain amino acid position 108, according to Kabat numbering.
  • polynucleotides or nucleic acids encoding the antigen binding proteins (including the multispecific antigen binding proteins) disclosed herein are provided, such as isolated polynucleotides or nucleic acids which are typically synthetic. Methods of making an antigen binding protein expressing these polynucleotides or nucleic acids are also provided.
  • polynucleotides encoding the antigen binding proteins disclosed herein are typically inserted in a cloning vector or into an expression vector for introduction into host cells that may be used to produce the desired quantity of the antigen binding proteins. Accordingly, in certain aspects, the invention provides expression vectors comprising the polynucleotides disclosed herein and host cells comprising these vectors and polynucleotides.
  • vectors used in accordance with the present invention as a vehicle for introducing into and expressing a desired gene in a cell.(e.g., polynucleotides or nucleic acids encoding the antigen binding proteins disclosed herein) in a cell.
  • a vector may be a self-replicating nucleic acid structure or incorporate into the genome upon introduction into the host cell.
  • vectors may readily be selected from the group consisting of plasmids, phages, viruses and retroviruses.
  • vectors compatible with the instant invention will comprise a selection marker, appropriate restriction sites to facilitate cloning of the desired gene and the ability to enter and/or replicate in eukaryotic or prokaryotic cells.
  • DNA elements which are derived from animal viruses such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses (e.g., RSV, MMTV, MOMLV or the like), or SV40 virus.
  • animal viruses such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses (e.g., RSV, MMTV, MOMLV or the like), or SV40 virus.
  • retroviruses e.g., RSV, MMTV, MOMLV or the like
  • SV40 virus retroviruses
  • Others involve the use of polycistronic systems with internal ribosome binding sites.
  • cells which have integrated the DNA into their chromosomes may be selected by introducing one or more markers which allow selection of transfected host cells.
  • the marker may provide for prototrophy to an auxotrophic host, biocide resistance (e.g., antibiotics) or resistance to heavy metals such as copper.
  • the selectable marker gene can either be directly linked to the DNA sequences to be expressed or introduced into the same cell by co-transformation. Additional elements may also be needed for optimal synthesis of mRNA. These elements may include signal sequences, splice signals, as well as transcriptional promoters, enhancers, and termination signals.
  • the cloned variable region genes are inserted into an expression vector along with the heavy and light chain constant region genes (e.g., human constant region genes) synthesized as discussed above.
  • the expression vector may be introduced into an appropriate host cell. That is, the host cells may be transformed.
  • Introduction of the plasmid into the host cell can be accomplished by various techniques well known to those of skill in the art. These include, but are not limited to, transfection (including electrophoresis and electroporation), protoplast fusion, calcium phosphate precipitation, cell fusion with enveloped DNA, microinjection, and infection with intact virus. See, Ridgway, A. A. G. “Mammalian Expression Vectors” Chapter 24.2, pp. 470-472 Vectors, Rodriguez and Denhardt, Eds.
  • Plasmid introduction into the host can be by electroporation.
  • the transformed cells are grown under conditions appropriate to the production of the light chains and heavy chains, and assayed for heavy and/or light chain protein synthesis.
  • Exemplary assay techniques include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence-activated cell sorter analysis (FACS), immunohistochemistry and the like.
  • the term “transformation” shall be used in a broad sense to refer to the introduction of DNA into a recipient host cell that changes the genotype and consequently results in a change in the recipient cell.
  • “host cells” refers to cells that have been transformed with vectors constructed using recombinant DNA techniques and encoding at least one heterologous gene.
  • the terms “cell” and “cell culture” are used interchangeably to denote the source of antibody unless it is clearly specified otherwise. In other words, recovery of polypeptide from the “cells” may mean either from spun down whole cells, or from the cell culture containing both the medium and the suspended cells.
  • a host cell line used for antibody expression is of mammalian origin. Those skilled in the art can determine particular host cell lines which are best suited for the desired gene product to be expressed therein. Exemplary host cell lines include, but are not limited to, DG44 and DUXB11 (Chinese hamster ovary lines, DHFR minus), HELA (human cervical carcinoma), CV-1 (monkey kidney line), COS (a derivative of CV-1 with SV40 T antigen), R1610 (Chinese hamster fibroblast) BALBC/3T3 (mouse fibroblast), HAK (hamster kidney line), SP2/O (mouse myeloma), BFA-lclBPT (bovine endothelial cells), RAJI (human lymphocyte), 293 (human kidney) and the like.
  • DG44 and DUXB11 Choinese hamster ovary lines, DHFR minus
  • HELA human cervical carcinoma
  • CV-1 monkey kidney line
  • COS a derivative
  • the cell line provides for altered glycosylation, e.g., afucosylation, of the antibody expressed therefrom (e.g., PER.C6® (Crucell) or FUT8- knock-out CHO cell lines (Potelligent® cells) (Biowa, Princeton, N.J.)).
  • PER.C6® Crucell
  • FUT8- knock-out CHO cell lines Potelligent® cells
  • Host cell lines are typically available from commercial services, e.g., the American Tissue Culture Collection, or from published literature.
  • the host cell is not a cell within a human body.
  • Genes encoding the antigen binding proteins featured in the invention can also be expressed in non-mammalian cells such as bacteria or yeast or plant cells.
  • non-mammalian microorganisms such as bacteria can also be transformed, i.e., those capable of being grown in cultures or fermentation.
  • Bacteria which are susceptible to transformation, include members of the enterob acteriaceae, such as strains of Escherichia coli or Salmonella, Bacillaceae, such as Bacillus sublilis: Pneumococcus,' Streptococcus, and Haemophilus influenzae.
  • the proteins when expressed in bacteria, the proteins can become part of inclusion bodies. The proteins must be isolated, purified and then assembled into functional molecules.
  • eukaryotic microbes may also be used. Saccharomyces cerevisiae, or common baker’s yeast, is the most commonly used among eukaryotic microorganisms, although a number of other strains are commonly available.
  • Saccharomyces cerevisiae or common baker’s yeast
  • yeast is the most commonly used among eukaryotic microorganisms, although a number of other strains are commonly available.
  • the plasmid YRp7 for example (Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al., Gene, 7: 141 (1979); Tschemper et al., Gene, 10: 157 (1980)), is commonly used.
  • This plasmid already contains the TRP1 gene which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example ATCC No. 44076 or PEP4-1 (Jones, Genetics, 85: 12 (1977)).
  • the presence of the trpl lesion as a characteristic of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.
  • the disclosure provides a nucleic acid encoding the antigen binding proteins recited above, such as the bispecific antigen binding protein recited above, in particular an isolated nucleic acid.
  • the disclosure provides a vector comprising the nucleic acid recited above, in particular an expression vector or a viral vector.
  • the disclosure provides a host cell comprising the vector, e.g., the expression vector, or the nucleic acid recited above.
  • the disclosure provides a method of manufacturing the antigen binding proteins recited above, such as the bispecific antigen binding protein recited above, comprising the steps of:
  • a method of manufacturing a bispecific antigen binding protein es described above comprising the steps of:
  • a method of manufacturing a CAR-expressing cell as described above comprising the steps of:
  • the antigen binding proteins, including the multispecific antigen binding proteins, of the disclosure may be engineered or optimized.
  • “optimized” or “optimization” refers to the alteration of an antigen binding protein to improve one or more functional properties. Alteration includes, but is not limited to, deletions, substitutions, additions, and/or modifications of one or more amino acids within an antigen binding protein.
  • the term "functional property" is a property of an antigen binding protein for which an improvement (e.g., relative to a conventional antigen binding protein, such as an antibody) is desirable and/or advantageous to one of skill in the art, e.g., in order to improve the manufacturing properties or therapeutic efficacy of an antigen binding protein.
  • the functional property is stability (e.g., thermal stability).
  • the functional property is solubility (e.g., under cellular conditions).
  • the functional property is aggregation behavior.
  • the functional property is protein expression (e.g., in a prokaryotic cell).
  • the functional property is refolding behavior following inclusion body solubilization in a manufacturing process.
  • the functional property is not an improvement in antigen affinity.
  • the improvement of one or more functional properties has no substantial effect on the affinity of the antigen binding protein.
  • Alterations such as deletions, substitutions, and/or insertions, can be introduced into parental sequences by a variety of standard techniques known in the art, such as combinatorial chemistry, site-directed DNA mutagenesis, PCR-mediated and/or cassette mutagenesis, peptide/protein chemical synthesis, chemical reaction specifically modifying reactive groups in the parental binding member.
  • the variants so formed can be tested by routine methods for their chemical, biological, biophysical and/or biochemical properties, e.g., by the methods described elsewhere herein.
  • the substitution is a conservative amino acid substitution.
  • conservative substitution refers to replacing an amino acid with a replacement amino acid that is physically, biologically, chemically and/or functionally similar to the replacement amino acid, e.g., has a similar size, shape, electric charge and/or chemical properties, including the ability to form covalent or hydrogen bonds.
  • Non-conservative substitutions may lead to substantial changes, e.g., with respect to the charge, dipole moment, size, hydrophilicity, hydrophobicity or conformation of the antigen binding protein.
  • the antigen binding protein of the disclosure is an scFv and is optimized by identifying preferred amino acid residues to be substituted, deleted, and/or added at amino acid positions of interest (e.g., amino acid positions identified by comparing a database of scFv sequences having at least one desirable property, e.g., as selected with Quality Control (QC) assay, versus a database of mature antibody sequences, e.g., the Kabat database) in an antigen binding protein.
  • QC Quality Control
  • the disclosure further provides “enrichment/exclusion” methods for selecting a particular amino acid residue.
  • the disclosure provides methods of engineering antigen binding proteins (e.g., scFvs) by mutating particular framework amino acid positions identified using the “functional consensus” approach described herein.
  • the framework amino acid positions are mutated by substituting the existing amino acid residue by a residue which is found to be an "enriched” residue using the "enrichment/exclusion” analysis methods described herein.
  • the disclosure provides a method of identifying an amino acid position for mutation in a single chain antibody (scFv), the scFv having VH and VL amino acid sequences, the method comprising: a) entering the scFv VH, VL or VH and VL amino acid sequences into a database that comprises a multiplicity of antibody VH, VL or VH and VL amino acid sequences such that the scFv VH, VL or VH and VL amino acid sequences are aligned with the antibody VH, VL or VH and VL amino acid sequences of the database; b) comparing an amino acid position within the scFv VH or VL amino acid sequence with a corresponding position within the antibody VH or VL amino acid sequences of the database; c) determining whether the amino acid position within the scFv VH or VL amino acid sequence is occupied by an amino acid residue that is conserved at the corresponding position within the antibody VH or VL amino acid sequences of
  • the antigen binding protein may comprise an Fc domain which is modified such that it does not induce cytotoxic immune responses and/or does not activate complement. For example, one or more substitutions may be introduced into the Fc domain so that its ADCC/ADCP or CDC effector function is inactivated.
  • Such antigen binding protein has the advantage of increased half-life when compared to antibody fragments with a molecular weight below 60 kDa, without mediating cytotoxic immune responses.
  • the antigen binding protein of the disclosure in particular when in the scFv format, may comprise a variable heavy chain having a nonpolar amino acid at position 11, 89 and/or 108, according to Kabat numbering.
  • variable heavy chain comprises: leucine (L) or serine (S) at amino acid position 11, according to Kabat numbering; valine (V), serine (S), or threonine (T) at amino acid position 89, according to Kabat numbering; and/or leucine (L), serine (S), or threonine (T) amino acid position 108, according to Kabat numbering.
  • the antigen binding protein (such as the multispecific antigen binding protein described above) is chemically and/or biologically modified.
  • the antigen binding protein may be glycosylated, phosphorylated, hydroxylated, PEGylated, HESylated, PASylated, XTENylated, sulfated, labeled with dyes and/or radioisotopes, conjugated with enzymes and/or toxins, and/or Albumin binding or fusion technology.
  • any nucleic acid sequence, plasmid or vector and/or host cell described herein may be modified accordingly.
  • Such modification may for example be done to optimize pharmacokinetics, the water solubility or to lower side effects.
  • PEGylation, PASylation, XTENylation, HESylation and/or the fusion to serum albumin may be applied to slow down renal clearance, thereby increasing plasma half-life time of the antigen binding protein.
  • the antigen binding molecules of the disclosure are operably linked to human serum albumin.
  • a modification adds a different functionality to the antigen binding protein, for example, a detection label for diagnostics or a toxin to combat cancer cells even more efficiently.
  • the antigen binding proteins and other polypeptides provided herein undergo co- and post-translational modifications as known in the art.
  • post-translational modifications include, but are not limited to, disulfide bond formation, glycosylation, cyclization (such as e.g., N- terminal pyroglutamate formation), have a N-terminal or C-terminal residue removed or “clipped" (for example, C-terminal lysine residues are often removed during the manufacturing process), deamidation, isomerization, oxidation, glycation, acylation, fucosylation, peptide bond cleavage, non-reductible cross-linking, truncation, and/or have part or all of a signal sequence incompletely processed.
  • the CD3 antigen binding domain comprises an N-terminal truncation of 1 or more amino acids (e.g., a N-terminal truncation of 1, 2, 3, 4, or 5 amino acids). In certain embodiments, the CD3 antigen binding domain comprises a C-terminal truncation of 1 or more amino acids (e.g., a C-terminal truncation of 1, 2, 3, 4, or 5 amino acids). In certain embodiments, the N-terminal and/or C-terminal truncation is a truncation of the Fab-(scFv)2 of a heavy chain amino acid sequence of SEQ ID NO: 62 and a light chain amino acid sequence of SEQ ID NO: 63.
  • the N- terminal and/or C-terminal truncation is a truncation of the Fab-(scFv)2 of a heavy chain amino acid sequence of SEQ ID NO: 64 and a light chain amino acid sequence of SEQ ID NO: 65.
  • the N-terminal and/or C-terminal truncation is a truncation of the Fab-(scFv)2 of a heavy chain amino acid sequence of SEQ ID NO: 66 and a light chain amino acid sequence of SEQ ID NO: 67.
  • the Q at position 1 of the antigen binding proteins is removed which may prevent pyroglutamate formation and protects the protein to be clipped by proteases; in certain embodiments, this may lower the possibility of having different charge variants which may result challenging in the CMC process.
  • An N-terminal R removal may in certain embodiments result in a lower pre-existing antibody response.
  • the variant sequence of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, and/or SEQ ID NO: 67 comprises a pyroglutamate at amino acid position 1.
  • the light chain of the antigen binding protein comprises pyroglutamate (pE) instead of the N-terminal glutamate.
  • the antigen binding protein is glycosylated.
  • Glycosylation refers to a process that attaches carbohydrates to proteins. In biological systems, this process is performed enzymatically within the cell as a form of co- translational and/or post- translational modification.
  • a protein can also be chemically glycosylated.
  • the carbohydrates may be N-linked to a nitrogen of asparagine or arginine side-chains; O-linked to the hydroxy oxygen of serine, threonine, tyrosine, hydroxylysine, or hydroxyproline side-chains; employ xylose, fucose, mannose, and N- acetyl glucosamine attached to a phospho-serine; and/or adding mannose sugar to a tryptophan residue found in a specific recognition sequence.
  • Glycosylation patterns may, e.g., be controlled by choosing appropriate cell lines, culturing media, protein engineering manufacturing modes and process strategies (see., HOSSLER, P. Optimal and consistent protein glycosylation in mammalian cell culture. Glycobiology 2009, vol. 19, no. 9, p. 936-949.).
  • the glycosylation patterns of the antigen binding proteins described herein are modified to enhance ADCC and CDC effector function.
  • the antigen binding protein may be engineered to control or alter the glycosylation pattern, e.g., by deleting and/or adding of one or more glycosylation sites.
  • the creation of glycosylation sites can e.g., be accomplished by introducing the corresponding enzymatic recognition sequence into the amino acid sequence of the antigen binding protein.
  • the (multispecific) antigen binding protein comprises a pyroglutamate (pE, pyrGlu, pyre or pGlu) instead of the N-terminal glutamine or the N-terminal glutamate.
  • the light chain of the antigen binding protein comprises pyroglutamate (pE) instead of the N-terminal glutamine.
  • the light chain of the antigen binding protein comprises pyroglutamate (pE) instead of the N-terminal glutamate.
  • such pyroglutamate (pE) modification has no impact on the safety and/or efficacy of the (multispecific) antigen binding protein.
  • the N-terminal glutamine (Q) of a light chain disclosed herein is clipped, e.g., to prevent pyroglutamate formation.
  • HESylation utilizes hydroxy ethyl starch ("HES") derivatives.
  • HESylation utilizes hydroxy ethyl starch (“HES") derivatives.
  • HESylation the antigen binding protein is linked to conformationally disordered polypeptide sequences composed of the amino acids proline (P), alanine (A) and serine (S), and XTENylation employs a similar, intrinsically disordered XTEN-polypeptide.
  • the antigen binding protein (e.g., the multispecific antigen binding protein) is linked to or combined with a detectable label, a therapeutic agent or a PK modifying moiety.
  • the antigen binding protein can is labelled with or conjugated to a second moiety which attributes one or more ancillary functions to the antigen binding protein.
  • the second moiety may have an additional immunological effector function, be effective in drug targeting or useful for detection.
  • the second moiety can, e.g., be chemically linked or fused genetically to the antigen binding protein using known methods in the art.
  • label refers to any substance or ion which is indicative of the presence of the antigen binding protein when detected or measured by physical or chemical means, either directly or indirectly.
  • the label may be directly detectable by, without being limited to, light absorbance, fluorescence, reflectivity, light scatter, phosphorescence, or luminescence properties, molecules or ions detectable by their radioactive properties or molecules or ions detectable by their nuclear magnetic resonance or paramagnetic properties.
  • indirect detection include light absorbance or fluorescence; for example, various enzymes which cause appropriate substrates to convert, e.g., from nonlight absorbing to light absorbing molecules, or from non-fluorescent to fluorescent molecules.
  • a labelled antigen binding protein is particularly useful for in vitro and in vivo detection or diagnostic purposes.
  • an antigen binding protein labelled with a suitable radioisotope, enzyme, fluorophore or chromophore can be detected by radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), or flow cytometry-based single cell analysis (e.g., FACS analysis), respectively.
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunosorbent assay
  • FACS analysis flow cytometry-based single cell analysis
  • the nucleic acids and/or vectors disclosed herein can be labeled for detection or diagnostic purposes, e.g., using labelled fragments thereof as probes in hybridization assays.
  • Non-limiting examples of second moi eties include radioisotopes (35S, 32P, 14C, 18F, and/or 1251), apoenzymes, enzymes (e.g., alkaline phosphatase, horseradish peroxidase, beta-galactosidase and/or angiogenin), co-factors, peptide moieties (e.g., a HIS-tag), proteins (e.g.
  • lectin e.g., serum albumin
  • carbohydrates e.g., mannose-6-phosphate tags
  • fluorophores e.g., fluorescein isothiocyanate (FITC)
  • FITC fluorescein isothiocyanate
  • phycoerythrin green/blue/red or other fluorescent proteins
  • allophycocyanin APC
  • chromophores vitamins (e.g., biotin), chelators, antimetabolites (e.g., methotrexate), toxins (e.g. a cytotoxic drug, or a radiotoxin).
  • the invention relates to drug conjugates (in particular antibody-drug conjugates ADCs) comprising the antigen binding proteins described herein, e.g., a monovalent or a multispecific antigen binding protein described herein (e.g., an antibody), conjugated to a toxin which further enhances efficient killing of specific cells, such as e.g., MHC-displayed NTDNNLAVY (SEQ ID NO: 68) positive cells.
  • ADCs antibody-drug conjugates
  • the toxin moiety is typically a small molecular weight moiety, such as MMAE/MMAF, DM1, chaliceamicin, anthracycline toxins, taxol, gramicidin D and/or colchicine, which may be linked via a peptide linker to the antigen binding protein.
  • the ADC comprises a scFv comprising the amino acid sequence of SEQ ID NO: 37, SEQ ID NO: 38, and/or SEQ ID NO: 39, or a variant as described herein, In certain embodiments, said variant is at least about 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 37, SEQ ID NO: 38 and/or SEQ ID NO: 39, respectively.
  • the variant comprises the HCDR1 amino acid sequence of SSYYYMC (SEQ ID NO: 25), the HCDR2 amino acid sequence of CIYAGSSGSTYYASWAKG (SEQ ID NO: 26), the HCDR3 amino acid sequence of GAGYGNDGHSL (SEQ ID NO: 27), the LCDR1 amino acid sequence of QASENIYNSLA (SEQ ID NO: 28), the LCDR2 amino acid sequence of GASNLES (SEQ ID NO: 29), and/or the LCDR3 amino acid sequence of QSTYYGHDNVGGA (SEQ ID NO: 30).
  • the toxin may be conjugated non-site-specifically or site-specifically to the antigen binding protein.
  • Non-site-specific conjugation typically involves the use of chemical linkers, e.g., with maleimide functionality, that mediate conjugation to lysine or cysteine amino acid side chains of the antigen binding protein or to the amino-group of the N-terminus.
  • Site-specific conjugation may be achieved using chemical, chemo- enzymatic, or enzymatic conjugations known in the art, e.g., employing bifunctional linkers, bacterial transglutaminase or sortase enzymes, linkers allowing Pictet-Spengler chemistry on formyl-glycine forming enzyme modified antigen binding proteins, or glycan-remodeled antigen binding proteins.
  • antigen binding proteins of the disclosure such as the multispecific antigen binding protein described above
  • the route of administration of the antigen binding proteins of the current disclosure may e.g., be oral, parenteral, by inhalation, or topical.
  • parenteral as used herein includes intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal or vaginal administration.
  • topical as used herein includes, but is not limited to, administration with liquid or solution eye drops, emulsions (e.g., oil-in-water emulsions), suspensions, and ointments.
  • a form for administration would be a solution for injection.
  • a suitable pharmaceutical composition for injection may comprise a buffer (e.g., acetate, phosphate or citrate buffer), a surfactant (e.g., polysorbate), optionally a stabilizer agent (e.g., human albumin), etc.
  • a buffer e.g., acetate, phosphate or citrate buffer
  • a surfactant e.g., polysorbate
  • optionally a stabilizer agent e.g., human albumin
  • Effective doses of the compositions of the present disclosure, for the treatment of the related conditions vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic.
  • the patient is a human, but non-human mammals, including transgenic mammals, can also be treated.
  • Treatment dosages may be titrated using routine methods known to those of skill in the art to optimize safety and efficacy.
  • the antigen binding proteins of the present disclosure may be administered in a pharmaceutically effective amount for the in vivo treatment of mammalian disorders.
  • the disclosed antigen binding proteins will be formulated to facilitate administration and promote stability of the active agent.
  • an “effective amount” of an agent e.g., a pharmaceutical composition, refers to an amount effective to achieve the desired therapeutic or prophylactic result, at dosages and for periods of time as necessary.
  • compositions in accordance with the present disclosure typically include a pharmaceutically acceptable, non-toxic, sterile carrier such as physiological saline, nontoxic buffers, preservatives and the like.
  • a pharmaceutically effective amount of the antigen binding proteins shall be held to mean an amount sufficient to achieve effective binding to an antigen and to achieve a benefit, e.g., to ameliorate symptoms of a disease or disorder or to detect a substance or a cell.
  • the antigen binding proteins will typically be capable of interacting with selected immunoreactive antigens on neoplastic or immunoreactive cells and provide for an increase in the death of those cells.
  • the pharmaceutical compositions of the present disclosure may be administered in single or multiple doses to provide for a pharmaceutically effective amount of the modified binding polypeptide.
  • the antigen binding proteins of the disclosure may be administered to a human or other animal in accordance with the aforementioned methods of treatment in an amount sufficient to produce a therapeutic or prophylactic effect.
  • the antigen binding proteins of the disclosure can be administered to such human or other animal in a conventional dosage form prepared by combining the antigen binding proteins of the disclosure with a conventional pharmaceutically acceptable carrier or diluent according to known techniques. It will be recognized by one of skill in the art that the form and character of the pharmaceutically acceptable carrier or diluent is dictated by the amount of active ingredient with which it is to be combined, the route of administration and other well-known variables.
  • a cocktail comprising one or more species of antigen binding proteins described in the current disclosure may prove to be particularly effective.
  • the nucleic acids described herein, the vectors described herein, the host cell cells described herein (in particular the immune cells bearing a CAR) or the compositions described herein may be administered to a human or other animal in accordance with the methods of treatment described above in an amount sufficient to produce a therapeutic or prophylactic effect.
  • “Efficacy” or “zw vivo efficacy” as used herein refers to the response to a therapy by the pharmaceutical composition of the disclosure, using e.g., standardized response criteria.
  • the success or in vivo efficacy of the therapy using a pharmaceutical composition of the disclosure refers to the effectiveness of the composition for its intended purpose, i.e., the ability of the composition to cause its desired effect.
  • the in vivo efficacy may be monitored by established standard methods for the specific diseases. In addition, various disease specific clinical chemistry parameters and other established standard methods may be used.
  • the compounds and cells described herein are administered in combination with one or more different pharmaceutical compounds.
  • therapeutic use of the compounds and cells described herein may be in combination with one or more therapies selected from the group of antibody therapy, chemotherapy, cytokine therapy, dendritic cell therapy, gene therapy, hormone therapy, laser light therapy, radiation therapy or vaccine therapy.
  • an antigen binding protein comprising a Fab domain which binds a cell surface protein of an immune cell linked to a first and second pMHC antigen binding protein.
  • the methods may be used to treat patients having any tumor type in which at least some of the cancer cells express a target peptide as disclosed herein displayed on a pMHC, such as MHC-displayed NTDNNLAVY (SEQ ID NO: 68).
  • target peptide positive cancers or cancer cells can be assessed using any method known in the art, including, but are not limited to, detecting RNA expression levels or histological methods such as Immunohistochemistry (H4C).
  • the target pMHC binding domain specifically targets an MHC restricted peptide derived of a tumor antigen.
  • the target peptide positive cancer is a solid tumor and/or a hematological tumor, optionally wherein the cancer is selected from esophageal cancer, gastric adenocarcinoma, lung adenocarcinoma, and lung squamous cancer.
  • the upregulation of MHC-displayed NTDNNLAVY (SEQ ID NO: 68) expression on the cell surface is a biomarker for cancer.
  • the disclosure provides a method for killing a target cell comprising a major histocompatibility complex (MHC) presenting a neoantigen, the method comprising: a) contacting a plurality of cells comprising immune cells and the target cell with the antigen binding protein described herein, such as the multispecific antigen binding protein above, wherein said antigen binding protein specifically binds to the pMHC on the surface of the target cell and to CD3 on the surface of the immune cells; b) forming a specific binding complex through the antigen binding protein interactions with the target cells and the immune cells, thereby activating the immune cells; and c) killing the target cell with the activated immune cells.
  • MHC major histocompatibility complex
  • the disclosure provides a method of treating cancer comprising the step of administering the antigen binding protein described herein, the nucleic acid described herein, vectors described herein, the host cells described herein or the pharmaceutical composition described herein, to a patient in need thereof.
  • the aforementioned antigen binding proteins including antibody-drug conjugates), nucleic acids, vectors or host cells (in particular immune cells expressing CARs) or the vector, are useful as a medicament.
  • a medicament includes a therapeutically effective amount of a molecule or cell as provided herein.
  • a respective molecule or host cell can be used for the production of a medicament useful in the treatment of one or more disorders, in particular disorders or diseases consisting of MHC-displayed NTDNNLAVY (SEQ ID NO: 68).
  • a method of treating disorder or diseases consisting of MHC-displayed NTDNNLAVY (SEQ ID NO: 68) is provided.
  • the method includes the steps of administering a pharmaceutically effective amount of a molecule or host cell as described herein, in particular the antigen binding proteins or a CAR expressing cell, to a subject in need thereof.
  • the pharmaceutical composition described above which includes such pharmaceutically effective amount of the antigen binding protein, nucleic acid, vector or host cell (e.g., immune cell) is administered to the subject.
  • the medicament referred to above may be administered to a subject.
  • the disclosure provides a method of treating a MHC- displayed NTDNNLAVY (SEQ ID NO: 68) positive cancers, in a patient in need thereof comprising administering to the patient a therapeutically effective amount of the antigen binding protein described herein, such as the multispecific antigen binding protein described herein, the CAR described herein, the host cell (e.g., immune cell) described herein, or the pharmaceutical composition recited above.
  • a therapeutically effective amount of the antigen binding protein described herein such as the multispecific antigen binding protein described herein, the CAR described herein, the host cell (e.g., immune cell) described herein, or the pharmaceutical composition recited above.
  • patients eligible for treatment with an antagonist as described herein are selected based on RNA sequencing and/or immunohistochemistry (H4C), such as for detection of total target peptide.
  • H4C immunohistochemistry
  • the subject in need of treatment can be a human or a non-human animal.
  • the subject is diagnosed with an MHC-displayed NTDNNLAVY (SEQ ID NO: 68) related disorder or may acquire such a disorder.
  • the animal might be genetically engineered to develop MHC-displayed NTDNNLAVY (SEQ ID NO: 68) related disorder.
  • An animal may also be genetically engineered in such a way that it shows the characteristics of MHC-displayed NTDNNLAVY (SEQ ID NO: 68) related disease.
  • disorders or diseases consisting of MHC- displayed NTDNNLAVY (SEQ ID NO: 68) expression is cancer.
  • the cancer is a solid tumor and/or a hematological tumor, optionally wherein the cancer is selected from cervical cancer, pancreatic cancer (such as pancreatic adenocarcinoma (PAAD), cervical cancer (such as cervical squamous cell carcinoma (CESC)), esophageal cancer, gastric cancer such as gastric adenocarcinoma, lung cancer (such as lung adenocarcinoma (LU AD), lung squamous cancer or Non-small cell lung cancer (NSCLC) (e.g., non-squamous NSCLC)), or breast cancer such as triple-negative breast cancer.
  • pancreatic cancer such as pancreatic adenocarcinoma (PAAD), cervical cancer (such as cervical squamous cell carcinoma (CESC)
  • esophageal cancer gastric cancer such as gastric adenocarcinoma
  • An antigen binding protein as disclosed herein may be used for detection or diagnostic purposes in vivo and/or in vitro.
  • a wide range of immunoassays using antibodies for detecting the expression in specific cells or tissues are known to the skilled person.
  • an antigen binding protein connected to a detectable label such as biotin.
  • the described antigen binding proteins are useful for detecting the presence of a target peptide- MHC complex as described elsewhere herein in a sample.
  • the detection may be for quantitative or qualitative purposes.
  • the sample is preferably of biological origin, such as blood, urine, cerebrospinal fluid, biopsy, lymph and/or non-blood tissues.
  • a biological sample comprises a cell or tissue from a human patient.
  • the method includes contacting a biological sample with an antigen binding protein described herein, the CAR described herein, the immune cell described herein, the under conditions permissive for binding of the inhibitor to the target peptide-MHC and then detecting the inhibitor- target peptide- MHC and then detecting the inhibitor- target complex.
  • Such method may be an in vitro or in vivo method. In some embodiments, such method is performed to select subjects eligible for therapy with the antigen binding protein described herein.
  • the described antigen binding proteins are useful for detecting the presence of a target peptide- MHC complex as described elsewhere herein.
  • a method comprising the steps of:
  • kits comprising at least one nucleic acid library or antigen binding protein, such as an antibody, including the multispecific antigen binding protein, or the pharmaceutical composition as described herein, typically together with a packaged combination of reagents with instructions.
  • the kit includes a composition containing an effective amount of said antigen binding protein in unit dosage form.
  • Such kit may comprise a sterile container comprising the composition; nonlimiting examples of such containers include, without being limited to, vials, ampoules, bottles, tubes, syringes, blister-packs.
  • the composition is a pharmaceutical composition and the containers are made of a material suitable for holding medicaments.
  • the kit may comprise in a first container the antigen binding protein in lyophilized form and a second container with a diluent (e.g., sterile water) for reconstitution or dilution of the antigen binding protein.
  • a diluent e.g., sterile water
  • said diluent is a pharmaceutically acceptable diluent.
  • the kit is for diagnostic purposes and the antigen binding protein is formulated for diagnostic applications.
  • the kit is for therapeutic purposes and the antigen binding protein is formulated for therapeutic applications.
  • the kit will further comprise a separate sheet, pamphlet or card supplied in or with the container with instructions for use. If the kit is intended for pharmaceutical use, it may further comprise one or more of the following: information for administering the composition to a subject having a related disease or disorder and a dosage schedule, description of the therapeutic agent, precautions, warnings, indications, counter-indications, overdosage information and/or adverse reactions.
  • HLA-A*01 :01 positive tumor samples from non-small cell lung cancer (NSCLC) patients were used for immunoprecipitation using the pan-HLA (W6/32) antibody.
  • Peptides eluted from HLA complexes were processed for mass spectrometry analysis.
  • Heavy-labelled KK-LC-1 peptide was spiked into the samples to allow quantification based on established calibration curve. Copy number of KK-LC-1 peptide per cell was calculated based on tissue weight.
  • Three out of six tumor biopsy samples were positive for KK-LC-1 target peptide NTDNNLAVY (SEQ ID NO: 68) with up to 400 copies per cell identified ( Figure 1). The most similar human peptide PTS was identified in very high numbers in each sample.
  • Example 2 Identification of healthy tissue-displayed off-target peptides posing cross-reactivity risk
  • BLAST Basic Local Alignment Search Tool
  • NTDNNLAVY SEQ ID NO: 68
  • sCRAP database search was performed using the target sequence NTDNNLAVY (SEQ ID NO: 68) as a query dedicated to HLA-A*01 :01.
  • Peptide hits from different search tools were combined and send for an analysis to the normal tissue immunopeptidome database at Alithea Bio.
  • Possible off-target peptides have been selected based on their expression profile on healthy tissue and immune cells.
  • Several peptides such as ICE1 (NTDNLLTEY; SEQ ID NO: 76) and PTS (ETDNNIVVY; SEQ ID NO: 74) do have high similarity to the KK-LC1 target peptide and are expressed in healthy tissue.
  • Another peptide such as TBL3 (TADHNLLLY; SEQ ID NO: 78) has lower similarity to the KK- LC1 target but is highly expressed among many healthy tissues and immune cells.
  • FIG. 3 A list of peptides posing a cross reactivity risk due to high sequence similarity to KK-LC-1 is shown in Table 3.
  • Figure 2 and Figure 3 show the expression levels of peptides listed in Table 3 in healthy tissues and immune cells, respectively.
  • Table 3. List of endogenous physiologically relevant peptides with high sequence similarity to KK-LC-1 NTDNNLAVY (SEQ ID NO: 68).
  • Inclusion bodies were washed three times with TBS supplemented with 0.5 % LDAO and twice with TBS. Such prepared inclusion bodies were solubilized using 8 M urea, 100 mM Tris-HCl pH 8 buffer at ratio of 6 mL buffer per 1 g inclusion body pellet. Refolding and purification of the solubilized HLA-A*01 :01 extracellular domain (SEQ ID NO: 23), P2m (SEQ ID NO: 24) and KK-LC-1 peptide NTDNNLAVY (peptides & elephants, SEQ ID NO: 68) were performed essentially as described by Rodenko et al. (2006).
  • HLA-A*01 :01/KK-LC-l complex was assessed by SDS- PAGE and SE-HPLC.
  • Amino acid sequences of each pMHC component are recited in Table 4 Table 4 - Amino acid sequences of HLA-A*01 :01/KK-LC-l antigen components
  • Lymphocytes were isolated from lymph nodes and spleen of immunized animals to screen for B-cells that specifically recognize HLA-A*01 :01/KK-LC-l (SEQ ID NO: 68). Therefore, the secondary lymphoid organs were mechanically dissociated to extract cells and perform density gradient cell centrifugation for the recovery of living lymphocytes. Fluorescence-activated cell sorting (FACS) was applied to specifically isolate antibody-secreting B-cells that bind to HLA-A*01 :01/KK-LC-l (SEQ ID NO: 68). Immunofluorescence staining of lymphocytes was performed with antibodies or reagents detecting distinct cell type markers and fluorescently labelled pMHC’s.
  • FACS Fluorescence-activated cell sorting
  • HLA-A*01 :01 A pool of unrelated peptides loaded on HLA-A*01 :01 was used for the deselection of B-cells that recognize epitopes on the HLA backbone. In addition, a deselection against similar peptides loaded on HLA-A*01 :01 was performed to deplete the population of B-cells with low target peptide specificity (Table 5). Target selection, on the other hand, was performed with fluorescently labelled HLA-A*01 :01/KK-LC-l (SEQ ID NO: 68).
  • IxlO 8 lymphocytes were resuspended in 300 pl cold FACS-buffer (PBS pH 7.4, 1% FCS, 2 mM EDTA) containing the antibodies and reagents for immunofluorescence staining and incubated for 1 h at 4°C. After incubation the cells were washed twice with cold FACS-buffer and centrifuged at 300 g (5 min, 4°C). The cells were finally resuspended in four ml cold FACS-buffer and filtered through a 35 pm cell strainer FACS tube.
  • 300 pl cold FACS-buffer PBS pH 7.4, 1% FCS, 2 mM EDTA
  • Single B-cells were sorted with the CytoFlex SRT Cell Sorter (Beckman Coulter Life Sciences) into round-bottom 96-well plates containing 50’000 mitomycin C growth- arrested EL4-B5 feeder cells (Zubler et al., 1985) in 200 pl RPMI 1640 cultivation medium.
  • the sorted B-cells were cultivated for 7 days at 37°C and 5% CO2. More than 100 secreted IgGs from the B-cell supernatant were tested by ELISA for its binding to the target complex HLA-A*01 :01/KK-LC-l(SEQ ID NO: 68) and to similar peptides loaded on HLA-A*01:01. Signal ratio from the specific target binding to the unspecific binding was determined to identify hits binding specifically to the target.
  • RNA was extracted from the B-cells to generate cDNA via reverse transcription PCR.
  • a multiplex PCR with different primer sets was performed for each hit to recover VH, VK or VX sequences in separate reactions. PCR products were finally sequenced and reformatted as monovalent bispecific Fab-scFv molecules.
  • Monovalent bispecific antigen binding proteins in Fab-scFv format were expressed by transient co-transfection in HEK293-6E cells.
  • Cells were cultured in suspension using polyethylenimine (PEI 40kD linear).
  • PEI 40kD linear polyethylenimine
  • HEK293-6E cells were seeded at 1.7 x 106 cells/mL in Freestyle F17 medium supplemented with 2 mM L-Glutamine and 25ug/mL G418.
  • DNA and PEI were added separately to 50 pL/mL medium without supplement. Both fractions were mixed at 1 :2.5 DNA:PEI ratio, vortexed and rested for 15 minutes.
  • Biotin CAPture reagent (Cytiva) was immobilized on a Series S Sensor Chip CAP (Cytiva) and the biotinylated ligand HLA- A*01 :01/KK-LC-l (SEQ ID NO: 68) and control off-target peptide complexes with high sequence identity to KK-LC-1 peptide of SEQ ID NO: 68, i.e., HLA-A*01 :01/DSG3 (SEQ ID NO: 73), HLA-A*01 :01/PTS (SEQ ID NO: 74), HLA-A*01 :01/KDM7A (SEQ ID NO: 75, and HLA-A*01 :01/ICEl (SEQ ID NO: 76) were diluted in HPS-EP+ running buffer (TEKNOVA) and captured for 300 s at a flow rate of 2 pL/min at flow cell 2 resulting in a ligand capture level of >100
  • At least three consecutive analyte injections were performed at suitable concentrations applying the single-cycle-kinetics mode at a flow rate of 30 pL/min with an association time of 90 s and the dissociation time set to >400 s.
  • the chip surface was regenerated according to the manufacturer’s instructions.
  • the data was fit to a 1 : 1 Langmuir binding model to determine the association rate constant k a and dissociation rate constant ka and calculate the equilibrium dissociation constant KD (ka/k a ), also denoted as affinity, using the BiacoreTM Insight Evaluation software.
  • M2759 showed the most favorable binding profile with KD of 84 nM to HLA-A*01 :01/KK-LC-l (SEQ ID NO: 68) and no binding to the control off-target peptide complexes HLA-A*01 :01/DSG3 (SEQ ID NO: 73), HLA- A*01:01/PTS (SEQ ID NO: 74), HLA-A*01 :01/KDM7A (SEQ ID NO: 75), and HLA- A*01 :01/ICEl (SEQ ID NO: 76).
  • the anti-KK-LC-1 scFv of the most promising hit M2759 was humanized.
  • Human germlines IMGT_hVH_3_66 and IMGT_ hVK_l-5, displaying high sequence identity to VH and VL amino acid sequences of M2759 scFv were selected as CDR acceptor scaffolds.
  • Rabbit-originating framework region 4 was replaced by the IGHJ 1*01 and IGLJ2*01 human junction gene sequences in the VH and VL, respectively.
  • Antibody fragment constructs in Fab-scFv format monovalent for CD3 and KK-LC-1 binding were generated, using a humanized sp34 variant.
  • M2769 and M2883 comprised humanized variants of M2759 with varying number of rabbit-originating residues.
  • Example 8 Characterization of affinity and molecular recognition profile of humanized HLA-A*01:01/KK-LC-lxCD3 bispecific antigen binding proteins
  • each residue of the KK-LC-1 peptide NTDNNLAVY (SEQ ID NO: 68) was replaced with alanine (with exception of position 7, which was replaced by serine), arginine or aspartic acid (with exception of position 3, which was replaced by serine instead of aspartic acid).
  • the kinetic parameters for each peptide-MHC were determined by SPR, according to the previously described method. Ko-values for each tested peptide loaded on HLA-A*01 :01 are shown in Table 7.
  • M2883 displayed a very broad binding profile with at least 6 peptide residues showing relevance for binding.
  • Affinity of M2883 to the pMHC complex was drastically reduced (Ko-values increased) when amino acids on positions 5, 6, and 8 of the peptide were mutated to alanine and position 7 to serine; positions 5, 6, 7, 8, and 9 were mutated to arginine, and positions 4, 5, 6, and 7 were mutated to aspartate. Additionally, moderate affinity reduction with 2-4-fold Ko-value increase was observed when position 3 was mutated to alanine or serine, position 4 to alanine, position 8 to aspartate, and position 9 to alanine or aspartate. Affinity increase of about 4-fold was observed when position 4 was mutated to arginine. M2769 and M2759 showed moderate to drastic affinity reduction when positions 3, 4, 5, 6, 8 and 9 were mutated to alanine and position 7 to serine, showing similar behavior to M2883.
  • TAP-deficient T2 cells were transduced with a lentiviral vector encoding for HLA-A*01 :01 and beta-2 microglobulin and carrying the puromycin resistance gene (VectorBuilder). T2 cells were seeded in 6-well plates at 200,000 cells per well and transduced with lentiviral particles at MOI 10. Selection of HLA-A*01 :01 -expressing cells (T2A1) in puromycin-containing medium was initiated after 48h and continued for at least 2 weeks before cells were used in experiments.
  • T2A1 cells were incubated with serum-free RPMI1640 medium containing peptides KK-LC-1, TBL3, ICE1, PTPRC, FUS, PTS, VAV1, ZNF430, KDM7A, FAP, PDE10A, CHTOP, ROBO1, GPAM, DMXL2, ZBTB40 or DSG3 at a concentration of 10 pM overnight in 96 wells plates. Then, cells were washed in complete RPMI 1640 medium and co-incubated with PBMCs (E:T ratio of 5: 1) and varying concentrations of M2883 (range of 0.005 nM - 100 nM) for 24 h.
  • PBMCs E:T ratio of 5: 1
  • M2883 range of 0.005 nM - 100 nM
  • T cell activation was determined by quantification of IFN-gamma in the cell supernatants and is shown in Figure 4. Specific T cell activation was observed for M2883 in the presence of the KK-LC-1 peptide, while no activation was detected in the presence of any of the tested off-target peptides, indicating a very specific profile of M2883.
  • alanine, arginine and aspartic acid scan mutagenesis of the target KK-LC-1 peptide was also performed in antigen presenting T2A1 cells. Briefly, T2A1 cells were incubated with complete RPMI1640 medium containing peptides KK-LC-1 and the respective alanine, arginine and aspartic acid peptides (Table 7) at a concentration of 10 pM in 384 wells plates.
  • M2883 showed an excellent specificity profile with at least 6 amino acid positions of the native KK-LC-1 peptide relevant for T cell activation. This outcome was consistent with the M2883 molecular recognition profile previously determined by affinity measurements (SPR, Example 8).
  • Cancer cell killing mediated by M2883 was analyzed in a time-resolved manner using the IncuCyte S3 system. Briefly, HLA-A*01 :01-positive KK-LC-l-positive (NCI-H1703 and EKVX) and HLA-A*01:01 -positive KK-LC-1 -negative (SK-MEL-30 and PC-3) cancer cells were transduced with Nuclight Red lentivirus (Sartorius) to stably express the mKate2 fluorescent protein. Cancer cells were seeded at the density of 1.5* 10 3 cells per well in a sterile 384-well flat bottom adhesion tissue culture plate overnight at 37°C and 5% CO2 in an incubator.
  • Molecule M2883 was added at the indicated concentrations (range of 0.005 nM - 100 nM). PBMCs were added as effector cells to each well at an E:T ratio of 10: 1. The plate was imaged by fluorescent microscopy to monitor cell growth for 72 h. The degree of cell killing was quantified by comparing the fold growth ratio of fluorescent target cancer cells over time, relative to their number at time 0. As depicted in Figure 6, molecule M2883 showed a very specific cancer cell killing of the KK-LC-1 -positive cancer cells and no killing of the KK-LC-1 -negative cancer cells. Good correlation of potency and target copy number was observed.
  • HCMEC 147 Human Cardiac Microvascular Endothelial Cells
  • HBSMC 297 Human Bronchial Smooth Muscle Cells
  • HAEpC_645 Human Small Airway Epithelial Cells
  • HPMEC 770 Human Pulmonary Microvascular Endothelial Cells
  • Cells were prepared in assay medium (RPMI 1640 containing 10% FBS and 1 % penicillin-streptomycin) and plated at 20,000 cells per well in a volume of 50 pL assay medium.
  • PBMCs effector cells were plated at 100,000 cells per well in a volume of 50 pL assay medium.
  • Varying concentrations of compound M2883 (range of 0.05 nM to 100 nM) were added to the plated wells in 15 pL assay volume. The final assay medium was made up to 150 pL per well. All reactions were performed in duplicates. The plates were incubated for 24h at 37°C/5% CO2. Supernatants were collected and analyzed by human Granzyme B ELISA kit (MabTech) according to the manufacturer’s instructions. HLA-A*01 :01 -positive and KK-LC-1 -positive cell line NCI- H1703 served as a positive control. Corresponding results are depicted in Figure 7. M2883 showed no reactivity and therefore a safe profile on all four tested primary human cells.
  • M2883 was further tested on an extended selection of human primary cells, according to the above-described protocol.
  • Tested cells included human cardiac fibroblasts (HCF 722), normal human lung fibroblasts (NHLF 19232) and human aortic smooth muscle cells (HAoSMC_173).
  • Human pulmonary fibroblasts (HPF 646) served as an HLA-A*01 :01 -negative, KK-LC-1 -negative control and cancer cell line NCI-H1703 as an HLA-A*01 :01 -positive, KK-LC-1 -positive control.
  • HCF 722 human cardiac fibroblasts
  • NHLF 19232 normal human lung fibroblasts
  • HoSMC_173 human aortic smooth muscle cells
  • Human pulmonary fibroblasts (HPF 646) served as an HLA-A*01 :01 -negative, KK-LC-1 -negative control and cancer cell line NCI-H1703 as an HLA-A*01 :01 -positive
  • Granzyme B release was observed in fibroblast cells HCF 722 and NHLF 19232 when treated with high concentrations of M2883. Both reacting primary cells showed lower reactivity compared to the positive control NCI-H1703 with about 10- fold difference in EC50 value. HAoSMC_173 showed only minor and negligible reactivity at the highest tested concentration of M2883. HLA-A*01 :01 -negative fibroblast cells HPF 646 showed no reactivity when treated with M2883, suggesting that the observed reactivity in NHLF 19232 and HCF 722 could be caused by HLA-A*01 :01 specific off- target peptide.
  • Variants of M2883 were generated and tested for binding to HLA- A*01 :01 loaded with KK-LC-1, KK-LC-1 N4R and GPAM derived peptides. Binding affinity was determined by SPR, as described previously. M2883 variant with VL_E27K substitution was identified, exhibiting a substantial reduction in binding affinity to KK- LC-1 N4R (KD of 201 nM) and GPAM (no detectable binding), while retaining the parental-like binding affinity to the target KK-LC-1 peptide (KD of 36 nM). Further optimization of the M2883 VL E27K variant was performed to enhance humanization, stability and producibility. This included affinity maturation and optimization of framework regions.
  • M2883 VL E27K variant led to the development of M3591, which exhibited a significantly higher human germline sequence identity score of 87.9%, compared to 80.2% of the parental molecule M2883. This identity score was determined based on the percentage of amino acid sequence identity between the aligned regions and their corresponding germline sequences.
  • affinity maturation of M2883 VL E27K molecule was performed. Briefly, several phage display scFv libraries were constructed with targeted randomizations in CDRs. These libraries were designed to introduce targeted diversity while preserving the structural integrity of the scFv framework, facilitating the identification of high-affinity antigen binders.
  • the scFv libraries were synthesized and cloned into a phagemid vector upstream of a gene encoding the minor coat protein pill.
  • the phagemid libraries were then transformed into E. coli TGI cells and the number of colonies growing on the plates was used to determine the library diversity, assuring proper representation of variant sequences.
  • the resulting libraries were subjected to three rounds of biopanning with countersei ection against a mix of HLA-A*01 :01/KK-LC-l_N4R, GPAM and DCAF13, and selection against HLA-A*01 :01/KK-LC-l antigen. Libraries were screened for hits using a monoclonal phage ELISA after second and third round of panning.
  • Affinity matured and optimized M3903, the humanization intermediate M3591 and the parental M2883 were subjected to a characterization of binding affinity and biophysical properties.
  • a summary of key improvements in biophysical properties, binding affinity and specificity is presented in Table 10.
  • Affinity to the target antigen HLA-A*01 :01/KK-LC-l and the relevant off-target similar peptide antigen HLA- A*01 :01/GPAM was evaluated using previously described methods. With a KD of 1.8 nM, M3903 showed a 15 -fold increase in binding affinity to KK-LC-1, compared to the parental binder M2883.
  • M3903 also demonstrated improved specificity to the off-target similar peptide complex HLA-A*01 :01/GPAM, which contains a positively charged residue at position 4, a feature considered important for further enhancing specificity.
  • Specificity window between HLA-A*01 :01/KK-LC-l and HLA-A*01 :01/GPAM binding was significantly improved through engineering from approximately 6-fold in M2883 and about 40-fold in M3591 to over 260-fold in M3903.
  • Thermostability of M2883, M3591 and M3903 was measured using differential scanning fluorimetry (DSF). For this purpose, the samples were diluted to a concentration of 0.5 mg/ml. Temperature increased by 1°C / min from 20 - 95°C. Data were analyzed with the PR Panta Analysis (x64) software. Thermostability including onset of protein unfolding (Tonset), melting temperature T m and aggregation temperature (T agg ) was determined. In molecules consisting of several domains, such as bispecific compounds in Fab-scFv format, melting temperature corresponding to the individual domains can be determined. The Fab domain typically shows T m of >75 °C.
  • the T m of scFv relates to the first inflection point in the melting curve of the tested molecule.
  • the results are shown in Table 10.
  • M3903 showed superior thermostability, compared to both precursors and a very high melting temperature of 75.2 °C with only one inflection point.
  • Molecule cumulant radius (average size of the particles) of M2883, M3591 and M3903 was determined by dynamic light scattering (DLS) using the Prometheus Panta instrument. Briefly, samples were mixed and 0.1 pm filtered before measurement. Analysis was performed at 20°C and at the DLS laser power determined by the discovery scan of the instrument. Data was analyzed with the PR Panta Analysis (x64) software. The results are shown in Table 10.
  • M2883 had the largest cumulant radius (4.0 nm), followed by M3591 (3.9 nm) and M3903 (3.7 nm), indicating a slight but consistent decrease in hydrodynamic size following molecule engineering. This trend suggests that M3903 could have a more compact structure and/or reduced self-association compared to the parental variants - both desirable qualities in therapeutic biologies.
  • M3903 was additionally tested for binding to the HLA-A*01 :01 complexes loaded with off-target peptides of physiological relevance and high sequence identity to KK-LC-1 peptide.
  • M3643 was tested for binding to said off-target peptides.
  • Tested ligands included HLA-A*01 :01/DSG3, HLA-A*01 :01/PTS, HLA- A*01:01/KDM7A, HLA-A*01:01/ICEl, HLA-A*01 :01/ROBOl, HLA-A*01:01/TBL3 and a positive control HLA-A*01 :01/KK-LC-l.
  • Binding affinity was determined by SPR using a previously described protocol. The resulting KD values are shown in Table 11. No binding or only low affinity interaction with at least 17-fold difference in binding affinity compared to the target HLA-A*01 :01/KK-LC-l was detected for the tested off-target peptide complexes for M3903 and M3643.
  • M3903 Safety of M3903 was evaluated in a primary cell assay, essentially as described in Example 11, and compared to the parental molecule M2883.
  • Tested cells included human lung fibroblasts (NHLF 19232), human aortic smooth muscle cells (HAoSMC_173) and the HLA-A*01 :01 -positive, KK-LC-1 -positive control cancer cell line NCI-H1703.
  • M3903 showed higher potency on the antigen-positive NCI-H1703 cells than the parental M2883 and no reactivity on the tested primary cells. This demonstrates not only improved efficacy but also greater specificity and a favorable safety profile of M3903.
  • M3904 was created by reformatting of M3903 into a bivalent bispecific T cell engager in a Fab-(scFv)2 format with anti-CD3 Fab and two anti-HLA- A*01 :01/KK-LC-l scFvs.
  • the Fab-(scFv)2 M3748 was created from M3741 and M3864 from M3643. This format enables efficient avidity-driven targeting of antigenexpressing cancer cells. Furthermore, with a size of 100 kDa, it is expected to exhibit a favorable pharmacokinetic profile, potentially supporting Q2W or Q3W dosing.
  • Bivalent bispecifics were produced using stable CHO pools in 14-day fed-batch cultivations.
  • Target protein was purified from clarified, sterile-filtered culture supernatants via affinity chromatography and cation exchange chromatography. Final protein purity was evaluated using SE-HPLC, CEX HPLC, and HIC HPLC ( Figure 9). All molecules showed a good manufacturability profile with titers of 3.8 g/L, 2.7 g/L and 2.8 g/L for M3904, M3748 and M3864, respectively, and a high product quality.
  • M3904 displayed a more efficient killing of both tested antigen-positive cell lines and a good correlation between potency and target copy number.
  • M3748 showed potency comparable to M2883 on antigen-positive cancer cells, and M3864 was less potent.
  • M3904-mediated T cell activation assessed by cytokine and cytolytic molecule release was determined in a coculture cellular assay.
  • HLA-A*01 flpositive and KK-LC-1 -positive cell line NCI-H1703 and HLA-A*01 :01-positive and KK- LC-1 -negative cell line SK-MEL-30 were prepared in assay medium (RPMI 1640 containing 10% FBS and 1 % penicillin-streptomycin) and plated at 20,000 cells per well in a volume of 100 pL assay medium. The following day, the medium was aspirated and PBMCs effector cells were plated at 100,000 cells per well in a volume of 90 pL assay medium.
  • Varying concentrations of compound M3904 were added to the plated wells in 10 pL assay volume. The final assay medium was topped up to 100 pL per well. All reactions were performed in duplicates. The plates were incubated for 24h at 37°C/5% CO2. Supernatants were collected and fFNy, IL-2, IL-6, TNFa, Granzyme B and IL- 10 were analyzed with MSD U-plex plates (Meso Scale Discovery) according to the manufacturer’s instructions.
  • IFNy, IL-2, IL-6, TNFa, Granzyme B, and IL-10 were detected only in NCI-H1703 co-culture supernatants following M3904 treatment, as shown in Figure 11, indicating a specific and robust activation, and functional response of T cells in vitro.
  • M3904-mediated T cell activation markers were analyzed in a coculture cellular assay. Briefly, HLA-A*01 :01 -positive and KK-LC-1 -positive cells NCI-H1703 were prepared in assay medium (RPMI 1640 containing 10% FBS and 1 % penicillinstreptomycin) and plated at 20,000 cells per well in a volume of 50 pL assay medium. PBMCs effector cells were plated at 100,000 cells per well in a volume of 50 pL assay medium. Varying concentrations of compound M3904 (range of 0.02 nM to 50 nM) were added to the plated wells in 15 pL assay volume.
  • the final assay medium was topped up to 150 pL per well. All reactions were performed in duplicates. Cells were incubated at 37°C/5% CO2. Analysis was performed at 48h and 96h time-points for different activation markers. After 48h, cells were collected and stained with Live/dead Fixable NIR and with the antibodies against CD3, CD4, CD8, together with CD69 and CD25 as activation markers. Samples were then fixed and stored at 4°C until analyzed by flow cytometry using the MACSQuant Analyzer 10. Analysis of T cell populations and activation markers was performed using the FlowLogic software. After 96h, cells were collected, permeabilized and stained with Live/dead Fixable NIR, together with antibodies against CD3, CD4, CD8 and Ki67 proliferation marker.
  • M3904 was tested for safety in a primary cell assay against an extended panel of HLA-A*01 :01 -positive human primary cells originating from critical tissues, namely lung, vascular system, heart and kidneys. Assays were performed essentially as described in Example 11. Tested cells included tracheal smooth muscle cells, bronchial smooth muscle cells, small airway epithelial cells, lung fibroblasts, pulmonary microvascular endothelial cells, aortic smooth muscle cells, aortic endothelial cells, cardiomyocytes, cardiac microvascular endothelial cells, renal cortical epithelial cells and renal epithelial cells.
  • HLA-A*01 :01 -positive and KK-LC-1 -positive cell line NCI-H1703 served as a positive control.
  • M3748 and M3864 were tested for safety in a primary cell assay against HLA-A*01 :01 -positive human primary cells human cardiac microvascular endothelial cells (HMVEC-C 75839), normal human lung fibroblasts (NHLF 19232), human cardiac microvascular endothelial cells (HCMEC 147), human pulmonary microvascular endothelial cells (HPMEC 770), bronchial smooth muscle cells (HBSMC 297) and aortic endothelial cells (HAEC 22171).
  • HMVEC-C 75839 normal human lung fibroblasts
  • HCMEC 147 human cardiac microvascular endothelial cells
  • HPMEC 770 human pulmonary microvascular endothelial cells
  • HBSMC 297 bronchial smooth muscle cells
  • HAEC 22171 aortic endothelial cells
  • HLA-A*01 :01 -positive and KK-LC-1 -positive cell line NCI-H1703 served as a positive control.
  • Corresponding results are shown in Figure 13B for M3748 and Figure 13C for M3864.
  • Low levels of Granzyme B were detected at the highest tested M3904 concentrations in lung fibroblasts and in renal epithelial cells, while all other tested primary cells showed no or negligible Granzyme B release at any tested M3904 concentration.
  • M3748 showed minor reactivity on lung fibroblasts at the highest tested compound concentrations, while M3864 did not elicit any Granzyme B release on any of the tested primary cells.
  • the newly identified relevant peptides included KDM7A (SEQ ID NO: 75), SRP72 (SEQ ID NO: 135, PTPN7 (SEQ ID NO: 136), ESYT1 (SEQ ID NO: 137), WDR81 (SEQ ID NO: 138), and SLC25A10 (SEQ ID NO: 139). These and the previously identified endogenous physiologically relevant peptides with high sequence similarity to KK-LC-1 (Table 3) were tested in a cytolytic molecule release assay using the peptide-pulsed T2A1 cells, essentially as described in Example 9. KK-LC-1 peptide served as a positive control. Resulting data are presented in Figure 15.
  • M3904 exhibited a highly specific profile, characterized by high GrzB release in the presence of the KK-LC-1 peptide. Only at high concentrations M3904 triggered minor GrzB release with the off-target peptides KDM7A, SRP72, PTPN7, ESYT1, WDR81, and SLC25A10. Among these, KDM7A induced the highest GrzB release, though it still maintained a large safety margin compared to the target KK-LC-1 peptide. No Granzyme B release was detected in the presence of other tested off-target peptides, indicating a very specific profile of M3904. [0507] The developability profile of M3904, M3748 and M3864 was assessed.

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Abstract

L'invention concerne des protéines de liaison à l'antigène ciblant des complexes (pCMH) peptide dérivé de KK-LC-1-CMH. Sont également décrites des protéines de liaison à un antigène multispécifiques comprenant un domaine de liaison à l'antigène ayant une spécificité à CD3, et au moins un domaine de liaison à un peptide cible. L'invention concerne également des méthodes de traitement associées.
PCT/EP2025/059178 2024-04-04 2025-04-03 Protéines de liaison à l'antigène ciblant un peptide kk-lc-1 à restriction hla Pending WO2025210181A1 (fr)

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