WO2025237420A1

WO2025237420A1 - Her2 targetting antibodies, chimeric antigen receptors, and uses thereof

Info

Publication number: WO2025237420A1
Application number: PCT/CN2025/095640
Authority: WO
Inventors: Yangbing Zhao; Xiaojun Liu; Jie Wang
Original assignee: UTC Therapeutics Shanghai Co Ltd
Current assignee: UTC Therapeutics Shanghai Co Ltd
Priority date: 2024-05-17
Filing date: 2025-05-19
Publication date: 2025-11-20
Anticipated expiration: 2026-11-17

Abstract

Disclosed herein are anti-HER2 antibodies and antigen-binding fragments, chimeric antigen receptors ("CARs") having these anti-HER2 antibodies and antigen-binding fragments ("HER2 CARs") and genetically modified immune effector cells having such HER2 CARs. Polynucleotides encoding the anti-HER2 antibodies and antigen-binding fragments and HER2 CARs are also provided herein. Compositions comprising anti-HER2 antibodies and antigen-binding fragments and HER2 CARs are also provided herein. The present disclosure also provides use of the anti-HER2 antibodies and antigen-binding fragments and genetically modified immune effector cells having such HER2 CARs in cancer treatment.

Description

HER2 TARGETTING ANTIBODIES, CHIMERIC ANTIGEN RECEPTORS, AND USES THEREOF

1. Field

The present application relates to molecular biology, cell biology, and immuno-oncology. In particular, provided herein include anti-HER2 antibodies, chimeric antigen receptors (CARs) comprising such anti-HER2 antibodies ( “HER2 CARs” ) , genetically engineered immune effector cells expressing such HER2 CARs, and uses thereof in treating tumors or cancers.

2. Background

Cell-based immunotherapy is a therapy with curative potential for the treatment of cancer. T cells and other immune cells can be modified to target tumor antigens through the introduction of genetic material coding for artificial or synthetic receptors for antigen, such as Chimeric Antigen Receptors (CARs) , specific to selected antigens. Targeted T cell therapy using CARs (CARTs) has shown recent clinical success in treating some hematologic malignancies. However, translating CAR-expressing T cell therapy to solid tumors poses several obstacles that must be overcome to achieve clinical benefit. Accordingly, there are needs for novel therapeutic strategies to design CARs for treating cancers, particularly, solid tumors, which strategies capable of inducing potent tumor eradication with minimal toxicity and immunogenicity. HER2 is expressed in a variety of human cancers, such as breast cancer, gastric cancer, ovarian cancer, cervical cancer, urothelial cancer, esophageal cancer, bladder cancer, colorectal cancer, endometrial cancer, kidney cancer, lung cancer, pancreatic cancer, head and neck cancer, sarcoma, glioblastoma, prostate cancer, and/or thyroid cancer.

Current therapies targeting HER2, however, have only had limited success. Thus, additional HER2-targeting therapeutic options represent unmet needs. The compositions and methods provided herein meet these needs and provide other relative advantages.

3. Summary

The present disclosure provides antibodies or antigen-binding fragments thereof that specifically bind HER2 (e.g., human HER2) , comprising: (a) a light chain variable region (VL) comprising a light chain CDR1 (LCDR1) , a light chain CDR2 (LCDR2) and a light chain CDR3 (LCDR3) have amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively; or a variant thereof having up to about 5 amino acid substitutions, additions, and/or deletions in the LCDRs; and/or (b) a heavy chain variable region (VH) comprising a heavy chain CDR1 (HCDR1) , a heavy chain CDR2 (HCDR2) , and a heavy chain CDR3 (HCDR3) have amino acid sequences of SEQ ID NOs: 4, 5, and 6; or a variant thereof having up to about 5 amino acid substitutions, additions, and/or deletions in the HCDRs.

In some embodiments, the antibodies and antigen-binding fragments thereof provided herein comprise a LCDR1, a LCDR2, a LCDR3, a HCDR1, a HCDR2 and a HCDR3, wherein (a) the LCDR1, CDR2 and CDR3 have the amino acid sequences of SEQ ID NOs: 1, 2, and 3 respectively; and (b) the HCDR1, CDR2 and CDR3 have the amino acid sequences of SEQ ID NOs: 4, 5, and 6 respectively.

In some embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise: (a) a VL having at least 85%, at least 90%, at least 95%, at least 98%, or 100%sequence identity to an amino acid sequence of SEQ ID NO: 7; and/or (b) a VH having at least 85%, at least 90%, at least 95%, at least 98%, or 100%sequence identity to an amino acid sequence of SEQ ID NO: 8.

In some embodiments, the antibodies and antigen-binding fragments thereof provided herein comprise a VL and a VH, wherein the VL and VH have the amino acid sequences of SEQ ID NO: 7 and 8, respectively.

In some embodiments, the antibodies and antigen-binding fragments thereof provided herein are monoclonal antibodies or antigen-binding fragments.

In some embodiments, the antibodies and antigen-binding fragments thereof provided herein are selected from the group consisting of an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, and an IgG4 antibody.

In some embodiments, the antibodies and antigen-binding fragments thereof provided herein selected from the group consisting of a Fab, a Fab’ , a F (ab’ ) 2, a Fv, a scFv, a (scFv) 2, a single domain antibody (sdAb) , and a heavy chain antibody (HCAb) .

In some embodiments, the antibody and antigen-binding fragment thereof provided herein is a scFv having the amino acid sequence of SEQ ID NO: 9.

Also provided herein are Chimeric Antigen Receptors (CARs) that specifically bind HER2, comprising, from N-terminus to C-terminus: (a) a HER2-binding domain that comprises the antibody or antigen-binding fragment thereof provided herein; (b) a transmembrane domain; and (c) a cytoplasmic domain.

In some embodiments of the CARs provide herein, the transmembrane domain is derived from CD8, CD28, CD3ζ, CD4, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, BTLA, TCR α chain, TCR β chain, or TCR ζ chain, CD3ε, CD45, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, or CD154.

In some embodiments of the CARs provide herein, the transmembrane domain comprises CD8 transmembrane region.

In some embodiments of the CARs provide herein, the transmembrane domain is CD8 transmembrane region having the amino acid sequence of SEQ ID NO: 15 and nucleotide sequence of SEQ ID NO: 16.

In some embodiments of the CARs provide herein, the cytoplasmic domain comprises a signaling domain derived from CD3ζ, FcRγ, FcγRIIa, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, DAP10, DAP12, or any combination thereof.

In some embodiments of the CARs provide herein, the cytoplasmic domain further comprises a co-stimulatory domain derived from CD28, 4-1BB (CD137) , OX40, ICOS, DAP10, 2B4, CD27, CD30, CD40, CD2, CD7, LIGHT, GITR, TLR, DR3, CD43, or any combination thereof.

In some embodiments of the CARs provide herein, the cytoplasmic domain comprises a CD3ζsignaling domain and a 4-1BB co-stimulatory domain. In some embodiments, the cytoplasmic domain is CD3ζ signaling domain having the amino acid sequence of SEQ ID NO: 19 and nucleotide sequence of SEQ ID NO: 20. In some embodiments, the co-stimulatory domain is 4-1BB having the amino acid sequence of SEQ ID NO: 17 and nucleotide sequence of SEQ ID NO: 18.

In some embodiments, the CARs provided herein further comprising a CD8 hinge domain between the HER2-binding domain and the transmembrane domain. In some embodiments, the hinge domain is CD8 hinge domain having the amino acid sequence of SEQ ID NO: 13 and nucleotide sequence of SEQ ID NO: 14.

In some embodiments, the CARs provide herein further comprising a signal peptide domain derived from CD8 at N-terminus. In some embodiments, the signal peptide domain is CD8 having the amino acid sequence of SEQ ID NO: 11 and nucleotide sequence of SEQ ID NO: 12.

In some embodiments, the CARs provide herein comprising an amino acid sequence of SEQ ID NO: 10.

Also provided herein are polynucleotides that encode the antibody or antigen-binding fragment and the CARs thereof provided herein. In some embodiments, the polynucleotide is a mRNA.

Also provided herein are vectors that comprise the polynucleotide provided herein.

Also provided herein are cells that comprise the CARs provided herein or the polynucleotide encoding the CARs provided herein.

In some embodiments, the cells provided herein are immune effector cells.

In some embodiments, the cells provided herein are derived from cells isolated from peripheral blood or bone marrow.

In some embodiments, the cells provided herein are derived from cells differentiated in vitro from stem or progenitor cells selected from the group consisting of a T cell progenitor cell, a hematopoietic stem and progenitor cell, a hematopoietic multipotent progenitor cell, an embryonic stem cell, and an induced pluripotent cell.

In some embodiments, the cells provided herein are T cells or NK cells.

In some embodiments, the cells provided herein are cytotoxic T cells, helper T cells, gamma delta Ts, CD4+/CD8+ double positive T cells, CD4+ T cell, a CD8+ T cells, CD4-/CD8-double negative T cells, CD3+ T cells, naive T cells, effector T cells, helper T cells, memory T cells, regulator T cells, Th0 cells, Th1 cells, Th2 cells, Th3 (Treg) cells, Th9 cells, Th17 cells, Thαβ helper cells, Tfh cells, stem memory TSCM cells, central memory TCM cells, effector memory TEM cells, or effector memory TEMRA cells.

In some embodiments, the cells provided herein recombinantly express a fusion protein comprising a first domain that activates an antigen-presenting cell (APC) and a second domain that activates an immune effector cell, wherein (i) the first domain comprises (a) a ligand that binds an activation receptor of the APC, or a receptor-binding fragment thereof, or (b) an antibody that binds an activation receptor of the APC, or an antigen-binding fragment thereof; and (ii) the second domain comprises (a) a co-stimulatory receptor of the immune effector cell, or a functional fragment thereof, (b) a co-stimulatory ligand of the immune effector cell, or a receptor-binding fragment thereof, or (c) an antibody that binds a co-stimulatory receptor of the immune effector cell, or an antigen-binding fragment thereof.

In some embodiments, the first domain comprises an anti-CD40 antibody or an antigen-binding fragment thereof. In some embodiments, the first domain comprises an anti-CD40 scFv. In some embodiments, the first domain comprises an anti-CD40 scFv having amino acid sequences of SEQ ID NOs: 38-49. In some embodiments, the second domain comprises a CD28 cytoplasmic domain. In some embodiments, the second domain comprises a CD28 cytoplasmic domain having amino acid sequences of SEQ ID NO: 36. In some embodiments, the second domain is an antibody or an antigen-binding fragment that binds CD28. In some embodiments, the second domain comprises a CD28 scFv. In some embodiments, the second domain comprises a CD28 scFv having an amino acid sequence of SEQ ID NO: 64.

In some embodiments, the N-terminus of the first domain of the fusion protein is linked to the C-terminus of the second domain of the fusion protein. In some embodiments, the N-terminus of the second domain of the fusion protein is linked to the C-terminus of the first domain of the fusion protein. In some embodiments, the first domain and the second domain of the fusion protein are linked via a linker. In some embodiments, the linkers have the amino acid sequence of (GGGGS) n, (n=1, 2, 3, 4, 5……) listed as SEQ ID NO: 21. In some embodiments, the linker are hinges derived from CD8, CD28 or lgG4m. In some embodiments, the linker is CD28 hinge having an amino acid sequence of SEQ ID NO: 25.

In some embodiments, the fusion protein is at least 85%, 90%, 95%, 98%, 99%or 100%identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 50-61 and 65-76. In some embodiments, the fusion protein comprises an amino acid sequence of SEQ ID NO: 55. In some embodiments, the fusion protein comprises an amino acid sequence of SEQ ID NO: 70. In some embodiments, the fusion protein comprises an amino acid sequence of SEQ ID NO: 61. In some embodiments, the fusion protein comprises an amino acid sequence of SEQ ID NO: 76.

In some embodiments, the cell further expresses a safety switch.

In some embodiments, the safety switch is expressed on the cell surface and comprises one or more monoclonal antibody-specific epitopes.

In some embodiments, the safety switch comprises, in sequence: a CD8α signal peptide, linker 1, a first monoclonal antibody-specific epitope, linker 2, a second monoclonal antibody-specific epitope, linker 3, a CD8 hinge region, and a CD8 transmembrane domain.

In some embodiments, the monoclonal antibody-specific epitopes are CD20 epitopes.

In some embodiments, the safety switch is co-expressed with the fusion protein comprising a first domain that activates an antigen-presenting cell (APC) and a second domain that activates an immune effector cell.

In some embodiments, the cell comprises an amino acid sequence as set forth in SEQ ID NO: 105 and/or a nucleotide sequence encoding the same.

Also provided herein are pharmaceutical compositions comprising the antibody or antigen-binding fragment provided herein, the CAR provided herein, the polynucleotide provided herein, the vector according provided herein and/or the cell of provided herein, and a pharmaceutically acceptable excipient.

Also provided herein are uses of the antibodies or antigen-binding fragments provided herein, the CARs provided herein, the polynucleotides provided herein, the vectors provided herein and/or the cells provided herein for the preparation of a medicament for the treatment of cancer.

Also provided herein are uses of the antibodies or antigen-binding fragments provided herein, the CARs provided herein, the polynucleotides provided herein, the vectors provided herein and/or the cells provided herein in cancer treatment.

Also provided herein are methods of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the antibodies or antigen-binding fragments provided herein, the CARs provided herein, the polynucleotides provided herein, the vectors provided herein and/or the cells provided herein.

In some embodiments of the methods provided herein, the cells or population of the cells is autologous to the subject.

In some embodiments, the methods provided herein further comprise obtaining cells from the subject.

In some embodiments, the methods provided herein further comprise administering an additional therapy to the subject.

In some embodiments of the methods provided herein, the subjects are human.

In some embodiments of the methods provided herein, the cancer is a HER2-expressing cancer.

In some embodiments of the methods provided herein, the cancer is a solid tumor or a hematological cancer.

In some embodiments of the methods provided herein, the cancer is breast cancer, gastric cancer, ovarian cancer, cervical cancer, urothelial cancer, esophageal cancer, bladder cancer, colorectal cancer, endometrial cancer, kidney cancer, lung cancer, pancreatic cancer, head and neck cancer, sarcoma, glioblastoma, prostate cancer, and/or thyroid cancer.

Also provided herein are methods of preparing a cell capable of expressing a CAR that specifically binds HER2, comprising transferring the polynucleotide provided herein into the cell.

In some embodiments of the method of preparing a cell capable of expressing a CAR, wherein the polynucleotide is transferred via electroporation.

In some embodiments of the method of preparing a cell capable of expressing a CAR, wherein the polynucleotide is transferred via viral transduction.

In some embodiments of the method of preparing a cell capable of expressing a CAR, wherein the method comprising using a lentivirus, a retrovirus, an adenovirus, or an adeno-associated virus for the viral transduction.

In some embodiments of the method of preparing a cell capable of expressing a CAR, wherein the polynucleotide is transferred using a transposon system.

In some embodiments of the method of preparing a cell capable of expressing a CAR, wherein the transposon system is Sleeping Beauty or PiggyBac.

In some embodiments of the method of preparing a cell capable of expressing a CAR, wherein the polynucleotide is transferred using gene-editing.

In some embodiments of the method of preparing a cell capable of expressing a CAR, wherein the polynucleotide is transferred using a CRISPR-Cas system, a ZFN system, or a TALEN system.

In some embodiments of the method of preparing a cell capable of expressing a CAR, wherein the cell is selected from the group consisting of a T cell, an NK cell, an NKT cell, a macrophage, a neutrophil, and a granulocyte cell.

Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

4. Brief Description of Drawings

FIG. 1. Cell phage ELISA screening results. The clones marked with a gray background (clone numbers 1-47) were selected as candidate clones for subsequent PCR-amplified scFv sequences for sequencing.

FIG. 2A. Flow cytometry results of the binding of anti-HER2 scFv that expressed in CART cells to HER2-Fc protein. FIG. 2B. Flow cytometry results of the binding of anti-HER2 scFv that expressed in CART cells to Biotin-SP-conjugated AffiniPure goat anti-mouse IgG- (Fab) 2 072 protein. FIG. 2C. Flow cytometry results of the binding of anti-HER2 scFv that expressed in CART cells to Biotin-SP-conjugated AffiniPure goat anti-human IgG- (Fab) 2 082 protein.

FIG. 3. The killing curves of mRNA-based H13 CART cells against SKOV3-CBG-GFP tumor cells at E/T ratio=3: 1 (FIG. 3A) and E/T ratio=1: 1 (FIG. 3B) , with the control of NO EP and other three anti-HER2 CART cells.

FIG. 4. The killing curves of mRNA-based anti-HER2 CART cells against A549-ESO-CBG-GFP tumor cells at E/T ratio=3: 1 (FIG. 4A) and E/T ratio=1: 1 (FIG. 4B) , with the control of NO EP and other three anti-HER2 CART cells, such as m4D5-3, m4D5-5 and m4D5.

FIG. 5. ELISA results. The IL-2 (FIG. 5A) and IFN-γ (FIG. 5B) results when mRNA-based anti-HER2 CART cells incubated with SKOV3-CBG-GFP and A549-ESO-CBG-GFP tumor cells at E/T ratio=1: 1.

FIG. 6A. pUTCK-H13 schematic diagram of lentiviral vector structure. FIG. 6B. pUTCK-H13. LACO schematic diagram of lentiviral vector structure. LACO includdes membrane LACOSTIM (mLACO) and soluble LACOSTIM (sLACO) .

FIG. 7. Anti-HER2 CAR-T and anti-HER2/LACOSTIM CAR-T cells, flow cytometry results stained with HER2-Fc antigen and anti-human IgG-Fc antibody. The staining results showed that the CAR molecules on anti-HER2 CAR-T and anti-HER2/LACOSTIM CAR-T cells were effectively expressed.

FIG. 8. Flow cytometry results of anti-HER2 CAR-T and anti-HER2/LACOSTIM CAR-T cells stained with CD40-Fc antigen and anti-human IgG-Fc antibody. The staining results showed that the expression level of LACOSTIM molecules on the CAR-T cells H13. A40517 expressing LACOSTIM on the cell membrane was very high. LACOSTIM molecules were not detected on the cell membrane of secreted LACOSTIM-expressing CAR-T cells H13.25-9.3h11, indicating that secreted LACOSTIM molecules may exist in soluble form outside the cells.

FIG. 9. Under the condition of E: T=3: 1 (FIG. 9A) , E: T=1: 1 (FIG. 9B) , and E: T=0.3: 1 (FIG. 9C) , the killing effect results of co-incubation of control NTD cells and 4 types of anti-HER2 CAR-T cells on SKOV3-CBG-GFP cells.

FIG. 10. Under the condition of E: T=3: 1 (FIG. 10A) , E: T=1: 1 (FIG. 10B) , and E: T=0.3: 1 (FIG. 10C) , the killing effect results of co-incubation of control NTD cells and 4 types of anti-HER2 CAR-T cells on A549-CBG-GFP cells. For A549 tumor cells with weak HER2 expression, the killing effect of H13, H13.25-9.3h11 and H13. A40517 was weaker than that of m4D5.

FIG. 11. Under the condition of E: T=3: 1, the killing effect results of co-incubation of control UTD cells and 4 types of anti-HER2 CAR-T cells on SY5Y cells, which had no expression of HER2 protein.

FIG. 12. pUTCK-R2. LACO schematic diagram of lentiviral vector structure. LACO here refers to soluble LACOSTIM (sLACO) . R here refers to mAb-specific epitope.

FIG. 13. Anti-HER2 CAR-T and anti-HER2/LACOSTIM CAR-T cells, flow cytometry results stained with HER2-Fc antigen and anti-human IgG-Fc antibody. The staining results showed that the CAR molecules on anti-HER2 CAR-T and anti-HER2/LACOSTIM CAR-T cells were effectively expressed.

FIG. 14. Flow cytometry results of anti-HER2/LACOSTIM CAR-T cells stained with CD40-Fc antigen and anti-human IgG-Fc antibody. The staining results showed that the LACOSTIM molecules on anti-HER2/LACOSTIM CAR-T cells were effectively expressed.

FIG. 15. Under the condition of E: T=3: 1, the killing effect results of co-incubation of control NTD cells and 5 types of anti-HER2 CAR-T cells on SKOV3-CBG-GFP cells.

FIG. 16. Under the condition of E: T=3: 1, the killing effect results of co-incubation of control NTD cells and 5 types of anti-HER2 CAR-T cells on A549-CBG-GFP cells. For A549 tumor cells with weak HER2 expression, the killing effect of H13 and H13+R2.25-9.3h11 was weaker than that of m4D5.

FIG. 17. Under the condition of E: T=3: 1, the killing effect results of co-incubation of control NTD cells and 5 types of anti-HER2 CAR-T cells on HepG2-CBG-GFP cells. For HepG2 tumor cells with weak HER2 expression, the killing effect of H13 and H13+R2.25-9.3h11 was weaker than that of m4D5.

FIG. 18. Under the condition of E: T=3: 1, the killing effect results of co-incubation of control NTD cells and 5 types of anti-HER2 CAR-T cells on Caski-CBG-GFP cells. For Caski tumor cells with weak HER2 expression, the killing effect of H13 and H13+R2.25-9.3h11 was weaker than that of m4D5.

FIG. 19. Under the condition of E: T=3: 1, the killing effect results of co-incubation of control NTD cells and 5 types of anti-HER2 CAR-T cells on SY5Y cells, which had no expression of HER2 protein.

FIG. 20A. The IL-2 results when lentivirus-transduced anti-HER2 CART cells incubated with SKOV3-CBG-GFP or A549-ESO-CBG-GFP tumor cells at E/T ratio=1: 1. FIG. 20B. The IFN-γresults when lentivirus-transduced anti-HER2 CART cells incubated with SKOV3-CBG-GFP or A549-ESO-CBG-GFP tumor cells at E/T ratio=1: 1.

FIG. 21A. Tumor size results after T cell injection in mice model. Compared with H13 group, the H13+R2.25-9.3h11 group could better control the tumor growth. FIG. 21B. Body weight of mice. FIG. 21C. The proportion of CD45+CD3+ cells in blood. H13+R2.25-9.3h11 group had a higher proportion of CART cells than H13.

5. Detailed Description

While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

The present disclosure provides novel antibodies, including antigen-binding fragments that specifically bind HER2 (e.g., human HER2) . Further, the present disclosure also provides chimeric antigen receptors (CARs) that comprise such antibodies or antigen-binding fragments that specifically bind HER2 (e.g., human HER2) , as well as engineered immune effector cells (e.g., T cells) and populations of cells that recombinantly express a CAR (e.g., CAR-Ts) that specifically binds HER2 (e.g., human HER2) . Pharmaceutical compositions comprising a therapeutically effective amount of such antibodies or antigen-binding fragments, and pharmaceutical compositions comprising a therapeutically effective amount of cells or population of cells are also disclosed herein. Also disclosed herein are uses of such pharmaceutical compositions for treating diseases and disorders relating to HER2 expression (e.g., HER2 expressing cancer) and related methods of treatment.
5.1 Definitions

Unless otherwise defined herein, scientific and technical terms used in the present disclosures shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art.

The present disclosure provides novel antibodies, including antigen-binding fragments that specifically bind HER2 (e.g., human HER2) . The term “HER2” includes any variants or isoforms of HER2 which are naturally expressed by cells. Accordingly, antibodies described herein can cross-react with HER2 from species other than human (e.g., cynomolgus HER2) . Alternatively, the antibodies can be specific for human HER2 and do not exhibit any cross-reactivity with other species. HER2 or any variants and isoforms thereof, can either be isolated from cells or tissues which naturally express them or be recombinantly produced using well-known techniques in the art and/or those described herein.

The term “antibody, ” and its grammatical equivalents as used herein refer to an immunoglobulin molecule that recognizes and specifically binds a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination of any of the foregoing, through at least one antigen-binding site wherein the antigen-binding site is usually within the variable region of the immunoglobulin molecule. As used herein, the term encompasses intact polyclonal antibodies, intact monoclonal antibodies, single-domain antibodies (sdAbs; e.g., camelid antibodies, alpaca antibodies) , single-chain Fv (scFv) antibodies, heavy chain antibodies (HCAbs) , light chain antibodies (LCAbs) , multispecific antibodies, bispecific antibodies, monospecific antibodies, monovalent antibodies, and any other modified immunoglobulin molecule comprising an antigen-binding site (e.g., dual variable domain immunoglobulin molecules) as long as the antibodies exhibit the desired biological activity. Antibodies also include, but are not limited to, mouse antibodies, camel antibodies, chimeric antibodies, humanized antibodies, and human antibodies. An antibody can be any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) , based on the identity of their heavy-chain constant domains referred to as alpha, delta, epsilon, gamma, and mu, respectively. Unless expressly indicated otherwise, the term “antibody” as used herein include “antigen-binding fragment” of intact antibodies. The term “antigen-binding fragment” as used herein refers to a portion or fragment of an intact antibody that is the antigenic determining variable region of an intact antibody. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F (ab’ ) 2, Fv, linear antibodies, single chain antibody molecules (e.g., scFv) , heavy chain antibodies (HCAbs) , light chain antibodies (LCAbs) , disulfide-linked scFv (dsscFv) , diabodies, tribodies, tetrabodies, minibodies, dual variable domain antibodies (DVD) , single variable domain antibodies (sdAbs; e.g., camelid antibodies, alpaca antibodies) , and single variable domain of heavy chain antibodies (VHH) , and bispecific or multispecific antibodies formed from antibody fragments.

The term “humanized antibody” as used herein refers to forms of non-human (e.g., murine) antibodies that are specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof that contain minimal non-human sequences. Typically, humanized antibodies are human immunoglobulin. In some instances, the Fv framework region residues of a human immunoglobulin are replaced with the corresponding residues in an antibody from a non-human species. In some instances, residues of the CDRs are replaced by residues from the CDRs of a non-human species (e.g., mouse, rat, hamster, camel) that have the desired specificity, affinity, and/or binding capability. The humanized antibody can be further modified by the substitution of additional residues either in the Fv framework region and/or within the replaced non-human residues to refine and optimize antibody specificity, affinity, and/or binding capability. The term “human antibody” as used herein refers to an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human made using any of the techniques known in the art.

The term “heavy chain” when used in reference to an antibody refers to a polypeptide chain of about 50-70 kDa, wherein the amino-terminal portion includes a variable region of about 120 to 130 or more amino acids and a carboxy-terminal portion that includes a constant region. The constant region can be one of five distinct types, referred to as alpha (a) , delta (δ) , epsilon (ε) , gamma (γ) and mu (μ) , based on the amino acid sequence of the heavy chain constant region. The distinct heavy chains differ in size: α, δ and γ contain approximately 450 amino acids, while μ and ε contain approximately 550 amino acids. When combined with a light chain, these distinct types of heavy chains give rise to five well known classes of antibodies, IgA, IgD, IgE, IgG and IgM, respectively, including four subclasses of IgG, namely IgGl, IgG2, IgG3 and IgG4. A heavy chain can be a human heavy chain.

The term “light chain” when used in reference to an antibody refers to a polypeptide chain of about 25 kDa, wherein the amino-terminal portion includes a variable region of about 100 to about 110 or more amino acids and a carboxy-terminal portion that includes a constant region. The approximate length of a light chain is 211 to 217 amino acids. There are two distinct types, referred to as kappa (κ) of lambda (λ) based on the amino acid sequence of the constant domains. Light chain amino acid sequences are well known in the art. A light chain can be a human light chain.

The term “variable domain” or “variable region” refers to a portion of the light or heavy chains of an antibody that is generally located at the amino-terminal of the light or heavy chain and has a length of about 120 to 130 amino acids in the heavy chain and about 100 to 110 amino acids in the light chain, and are used in the binding and specificity of each particular antibody for its particular antigen. The variable domains differ extensively in sequence between different antibodies. The variability in sequence is concentrated in the CDRs while the less variable portions in the variable domain are referred to as framework regions (FR) . The CDRs of the light and heavy chains are primarily responsible for the interaction of the antibody with antigen. Numbering of amino acid positions used herein is according to the EU Index, as in Kabat et al. (1991) Sequences of proteins of immunological interest. (U.S. Department of Health and Human Services, Washington, D.C. ) 5thed. A variable region can be a human variable region.

A CDR refers to one of three hypervariable regions (H1, H2 or H3) within the non-framework region of the immunoglobulin (Ig or antibody) VH β-sheet framework, or one of three hypervariable regions (L1, L2 or L3) within the non-framework region of the antibody VL β-sheet framework. Accordingly, CDRs are variable region sequences interspersed within the framework region sequences. CDR regions are well known to those skilled in the art and have been defined by a variety of methods/systems. These systems and/or definitions have been developed and refined over years and include Kabat, Chothia, IMGT, AbM, and Contact. For example, Kabat defines the regions of most hypervariability within the antibody variable (V) domains (Kabat et al, J. Biol. Chem. 252: 6609-6616 (1977) ; Kabat, Adv. Prot. Chem. 32: 1-75 (1978) ) . The Chothia definition is based on the location of the structural loop regions, which defines CDR region sequences as those residues that are not part of the conserved β-sheet framework, and thus are able to adapt different conformations (Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987) ) . Both terminologies are well recognized in the art. Additionally, the IMGT system is based on sequence variability and location within the structure of the variable regions. The AbM definition is a compromise between Kabat and Chothia. The Contact definition is based on analyses of the available antibody crystal structures. Software programs (e.g., abYsis) are available and known to those of skill in the art for analysis of antibody sequence and determination of CDRs. The positions of CDRs within a canonical antibody variable domain have been determined by comparison of numerous structures (Al-Lazikani et al, J. Mol. Biol. 273: 927-948 (1997) ; Morea et al, Methods 20: 267-279 (2000) ) . Because the number of residues within a hypervariable region varies in different antibodies, additional residues relative to the canonical positions are conventionally numbered with a, b, c and so forth next to the residue number in the canonical variable domain numbering scheme (Al-Lazikani et al., supra (1997) ) . Such nomenclature is similarly well known to those skilled in the art.

For example, CDRs defined according to either the Kabat (hypervariable) or Chothia (structural) designations, are set forth in the table below.

¹Residue numbering follows the nomenclature of Kabat et al., supra

²Residue numbering follows the nomenclature of Chothia et al., supra

One or more CDRs also can be incorporated into a molecule either covalently or noncovalently to make it an immunoadhesin. An immunoadhesin can incorporate the CDR (s) as part of a larger polypeptide chain, can covalently link the CDR (s) to another polypeptide chain, or can incorporate the CDR (s) noncovalently. The CDRs permit the immunoadhesin to bind to a particular antigen of interest. The CDR regions can be analyzed by, for example, abysis website.

Thus, unless otherwise specified, a CDR, or individual specified CDRs (e.g., LCDR1, LCDR2, LCDR3) , of a given antibody or region thereof, such as a variable region thereof, should be understood to encompass a (or the specific) complementary determining region as defined by any of the aforementioned schemes, or other known schemes. For example, where it is stated that a particular CDR (e.g., a HCDR3) contains the amino acid sequence of a corresponding CDR in a given VH or VL region amino acid sequence, it is understood that such a CDR has a sequence of the corresponding CDR (e.g., HCDR3) within the variable region, as defined by any of the aforementioned schemes, or other known schemes. In some embodiments, specific CDR sequences are specified, although it is understood that a provided antibody can include CDRs as described according to any of the other aforementioned numbering schemes or other numbering schemes known to a skilled artisan. Likewise, unless otherwise specified, a FR or individual specified FR (s) (e.g., VH FRl, VH FR2, VH FR3, VH FR4) , of a given antibody or region thereof, such as a variable region thereof, should be understood to encompass a (or the specific) framework region as defined by any of the known schemes.

The terms “epitope” and “antigenic determinant” are used interchangeably herein an refer to the site on the surface of a target molecule to which an antibody or antigen-binding fragment binds, such as a localized region on the surface of an antigen. The target molecule can comprise, a protein, a peptide, a nucleic acid, a carbohydrate, or a lipid. An epitope having immunogenic activity is a portion of a target molecule that elicits an immune response in an animal. An epitope of a target molecule having antigenic activity is a portion of the target molecule to which an antibody binds, as determined by any method well known in the art, including, for example, by an immunoassay. Antigenic epitopes need not necessarily be immunogenic. Epitopes often consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three-dimensional structural characteristics as well as specific charge characteristics. The term, “epitope” includes linear epitopes and conformational epitopes. A region of a target molecule (e.g., a polypeptide) contributing to an epitope can be contiguous amino acids of the polypeptide or the epitope can come together from two or more non-contiguous regions of the target molecule. The epitope may or may not be a three-dimensional surface feature of the target molecule. Epitopes formed from contiguous amino acids (also referred to as linear epitopes) are typically retained upon protein denaturing, whereas epitopes formed by tertiary folding (also referred to as conformational epitopes) are typically lost upon protein denaturing. An epitope typically includes at least 3, and more usually, at least 5, 6, 7, or 8-10 amino acids in a unique spatial conformation.

The term “specifically binds, ” as used herein, means that a polypeptide or molecule interacts more frequently, more rapidly, with greater duration, with greater affinity, or with some combination of the above to the epitope, protein, or target molecule than with alternative substances, including related and unrelated proteins. A binding moiety (e.g., antibody) that specifically binds a target molecule (e.g., antigen) can be identified, for example, by immunoassays, ELISAs, SPR (e.g., Biacore) , or other techniques known to those of skill in the art. Typically, a specific reaction will be at least twice background signal or noise and can be more than 10 times background. See, e.g., Paul, ed., 1989, Fundamental Immunology Second Edition, Raven Press, New York at pages 332-336 for a discussion regarding antibody specificity. A binding moiety that specifically binds a target molecule can bind the target molecule at a higher affinity than its affinity for a different molecule. In some embodiments, a binding moiety that specifically binds a target molecule can bind the target molecule with an affinity that is at least 20 times greater, at least 30 times greater, at least 40 times greater, at least 50 times greater, at least 60 times greater, at least 70 times greater, at least 80 times greater, at least 90 times greater, or at least 100 times greater, than its affinity for a different molecule. In some embodiments, a binding moiety that specifically binds a particular target molecule binds a different molecule at such a low affinity that binding cannot be detected using an assay described herein or otherwise known in the art. In some embodiments, “specifically binds” means, for instance, that a binding moiety binds a molecule target with a KD of about 0.1 mM or less. In some embodiments, “specifically binds” means that a polypeptide or molecule binds a target with a KD of at about 10 μM or less or about 1 μM or less. In some embodiments, “specifically binds” means that a polypeptide or molecule binds a target with a KD of at about 0.1 μM or less, about 0.01 μM or less, or about 1 nM or less. Because of the sequence identity between homologous proteins in different species, specific binding can include a polypeptide or molecule that recognizes a protein or target in more than one species. Likewise, because of homology within certain regions of polypeptide sequences of different proteins, specific binding can include a polypeptide or molecule that recognizes more than one protein or target. It is understood that, in some embodiments, a binding moiety (e.g., antibody) that specifically binds a first target may or may not specifically bind a second target. As such, “specific binding” does not necessarily require (although it can include) exclusive binding, i.e., binding to a single target. Thus, a binding moiety (e.g., antibody) can, in some embodiments, specifically bind more than one target. For example, an antibody can, in certain instances, comprise two identical antigen-binding sites, each of which specifically binds the same epitope on two or more proteins. In certain alternative embodiments, an antibody can be bispecific and comprise at least two antigen-binding sites with differing specificities.

The term “binding affinity” as used herein generally refers to the strength of the sum total of noncovalent interactions between a binding moiety and a target molecule (e.g., antigen) . The binding of a binding moiety and a target molecule is a reversible process, and the affinity of the binding is typically reported as an equilibrium dissociation constant (KD) . KD is the ratio of a dissociation rate (koff or kd) to the association rate (kon or ka) . The lower the KD of a binding pair, the higher the affinity. A variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present disclosure. Specific illustrative embodiments include the following. In some embodiments, the “KD” or “KD value” can be measured by assays known in the art, for example by a binding assay. The KD may be measured in a radiolabeled antigen binding assay (RIA) (Chen, et al., (1999) J. Mol Biol 293: 865-881) . The KD or KD value may also be measured by using surface plasmon resonance assays by Biacore, using, for example, a BIAcoreTM-2000 or a BIAcoreTM-3000 BIAcore, Inc., Piscataway, NJ) , or by biolayer interferometry using, for example, the OctetQK384 system (ForteBio, Menlo Park, CA) .

The term “variant” as used herein in relation to a protein or a polypeptide with particular sequence features (the “reference protein” or “reference polypeptide” ) refers to a different protein or polypeptide having one or more (such as, for example, about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, or about 1 to about 5) amino acid substitutions, deletions, and/or additions as compared to the reference protein or reference polypeptide. The changes to an amino acid sequence can be amino acid substitutions. The changes to an amino acid sequence can be conservative amino acid substitutions. A functional fragment or a functional variant of a protein or polypeptide maintains the basic structural and functional properties of the reference protein or polypeptide.

The terms “polypeptide, ” “peptide, ” “protein, ” and their grammatical equivalents as used interchangeably herein refer to polymers of amino acids of any length, which can be linear or branched. It can include unnatural or modified amino acids or be interrupted by non-amino acids. A polypeptide, peptide, or protein can also be modified with, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification.

The terms “polynucleotide, ” “nucleic acid, ” and their grammatical equivalents as used interchangeably herein mean polymers of nucleotides of any length and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase.

The terms “identical, ” percent “identity, ” and their grammatical equivalents as used herein in the context of two or more polynucleotides or polypeptides, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity. The percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to obtain alignments of amino acid or nucleotide sequences are well-known in the art. These include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and variants thereof. In some embodiments, two polynucleotides or polypeptides provided herein are substantially identical, meaning they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection. In some embodiments, identity exists over a region of the amino acid sequences that is at least about 10 residues, at least about 20 residues, at least about 40-60 residues, at least about 60-80 residues in length or any integral value there between. In some embodiments, identity exists over a longer region than 60-80 residues, such as at least about 80-100 residues, and in some embodiments the sequences are substantially identical over the full length of the sequences being compared, such as the coding region of a target protein or an antibody. In some embodiments, identity exists over a region of the nucleotide sequences that is at least about 10 bases, at least about 20 bases, at least about 40-60 bases, at least about 60-80 bases in length or any integral value there between. In some embodiments, identity exists over a longer region than 60-80 bases, such as at least about 80-1000 bases or more, and in some embodiments the sequences are substantially identical over the full length of the sequences being compared, such as a nucleotide sequence encoding a protein of interest.

The term “vector, ” and its grammatical equivalents as used herein refer to a vehicle that is used to carry genetic material (e.g., a polynucleotide sequence) , which can be introduced into a host cell, where it can be replicated and/or expressed. Vectors applicable for use include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes and artificial chromosomes, which can include selection sequences or markers operable for stable integration into a host cell’s chromosome. Additionally, the vectors can include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes that can be included, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media. Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like which are well known in the art. When two or more polynucleotides are to be co-expressed, both polynucleotides can be inserted, for example, into a single expression vector or in separate expression vectors. For single vector expression, the encoding polynucleotides can be operationally linked to one common expression control sequence or linked to different expression control sequences, such as one inducible promoter and one constitutive promoter. The introduction of polynucleotides into a host cell can be confirmed using methods well known in the art. It is understood by those skilled in the art that the polynucleotides are expressed in a sufficient amount to produce a desired product (e.g., an anti-HER2 antibody or antigen-binding fragment as described herein) , and it is further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art.

The term “genetic engineering” or its grammatical equivalents when used in reference to a cell is intended to mean alteration of the genetic materials of the cell that is not normally found in a naturally occurring cell. Genetic alterations include, for example, modifications introducing expressible polynucleotides, other additions, mutations/alterations, deletions and/or other functional disruption of the cell’s genes. Such modifications can be done in, for example, coding regions and functional fragments thereof of a gene. Additional modifications can be done in, for example, non-coding regulatory regions in which the modifications alter expression of a gene.

The term “transfer, ” “transduce, ” “transfect, ” and their grammatical equivalents as used herein refer to a process by which an exogenous polynucleotide is introduced into the host cell. A “transferred, ” “transfected, ” or “transduced” cell is one which has been transferred, transduced, or transfected with an exogenous polynucleotide. The cell includes the primary subject cell and its progeny. A polynucleotide can be “transferred” into a host cell using any type of approaches known in the art, including, e.g., a chemical method, a physical method, or a biological method. A polynucleotide is commonly “transduced” into a host cell using a virus. By contrast, a polynucleotide is commonly “transfected” into a host cell using a non-viral approach. These terms are used interchangeable at times, and a person of ordinary skill in the art would readily understand their meanings in different contexts.

As used herein, the term “encode” and its grammatical equivalents refer to the inherent property of specific sequences of nucleotides in a polynucleotide or a nucleic acid, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein. Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA can include introns.

A polypeptide, peptide, protein, antibody, polynucleotide, vector, cell, or composition which is “isolated” is a polypeptide, peptide, protein, antibody, polynucleotide, vector, cell, or composition which is in a form not found in nature. Isolated polypeptides, peptides, proteins, antibodies, polynucleotides, vectors, cells, or compositions include those which have been purified to a degree that they are no longer in a form in which they are found in nature. In some embodiments, a polypeptide, peptide, protein, antibody, polynucleotide, vector, cell, or composition which is isolated is substantially pure.

The term “immune effector cell” and its grammatical equivalents as used herein and understood in the art refer to cells that are of hematopoietic origin and play a direct role in the immune response against a target, such as a pathogen, a cancer cell, or a foreign substance. Immune effector cells include T cells, B cell, natural killer (NK) cells, NKT cells, macrophages, granulocytes, neutrophils, eosinophils, mast cells, and basophils.

“Stimulation” of an immune effector cell means a primary response induced by binding of a stimulatory molecule with its cognate ligand thereby mediating a signal transduction event in the immune effector cell which can alter expression of certain genes and/or reorganization of cytoskeletal structures, and the like. A “stimulatory molecule” of an immune effector cell refers to a molecule on the immune effector cell that, upon binding with its cognate ligand, which is commonly present on an APC, can mediate signal transduction to promote the maturation, differentiation, proliferation, and/or activation of the immune effector cell. For example, a stimulatory molecule of the T cells, the TCR/CD3 complex triggers the activation of the T cells. The ligand for a stimulatory molecule, or “stimulatory ligand, ” means a ligand that is commonly present on an APC and can bind with a stimulatory molecule on the immune effector cell to mediate a primary response by the immune effector cell, including, but not limited to, maturation, differentiation, activation, initiation of an immune response, proliferation, and the like. Stimulatory ligands are well-known in the art and encompass, for example, an MHC Class I molecule loaded with a peptide, an anti-CD3 antibody, a superagonist anti-CD28 antibody, and a superagonist anti-CD2 antibody.

A “co-stimulatory signal, ” as used herein and understood in the art, refers to a signal from a co-stimulatory receptor (e.g., CD28 or 4-1BB) , which in combination with a primary signal (e.g., TCR/CD3) promotes optimal clonal expansion, differentiation and effector functions of immune effector cells (e.g., T cells) . A “co-stimulatory receptor” of an immune effector cell, s used herein and understood in the art, refers to a molecule on the immune effector cell that specifically binds with a “co-stimulatory ligand” to mediate a co-stimulatory response by the immune effector cell, such as heightened activation or proliferation of the immune effector cell. Co-stimulatory receptors for immune effector cells include, but are not limited to, CD28, 4-1BB, ICOS, CD27, OX40, DAP10, CD30, 2B4, CD2, LIGHT, GITR, TLR, DR3, and CD43. A “functional fragment” of a co-stimulatory receptor is a fragment of the co-stimulatory receptor that retains its function to mediate a co-stimulatory signal and stimulate the immune effector cell. In some embodiments, a functional fragment of a co-stimulatory receptor retains the co-stimulatory domain of the co-stimulatory receptor. In some embodiments, the co-stimulatory domain is the cytoplasmic domain of the co-stimulatory receptor. In some embodiments, signals from co-stimulatory receptors of immune effector cells (e.g., T cells) lower the activation threshold for the immune effector cells. In some embodiments, signals from co-stimulatory receptors of T cells lead to the augmentation of TCR signaling events necessary for efficient cytokine production (via augmented transcriptional activity and messenger RNA stabilization) , cell cycle progression, survival, regulation of metabolism and T cell responses.

A “co-stimulatory ligand, ” as used herein and understood in the art, refers to a molecule that specifically binds a cognate co-stimulatory receptor on an immune effector cell, thereby providing a signal which, in addition to the primary signal provided by the stimulatory molecule, mediates a response in the immune effector cell, including, but not limited to, proliferation, activation, differentiation, and the like. The co-stimulatory ligand can be present on an APC (e.g., a dendritic cell) . Co-stimulatory ligands include, but are not limited to, CD58, HER2, CD83, CD80, CD86, CD137L (4-1BBL) , CD252 (OX40L) , CD275 (ICOS-L) , CD54 (ICAM-1) , CD49a, CD112 (PVRL2) , CD150 (SLAM) , CD155 (PVR) , CD265 (RANK) , CD270 (HVEM) , TL1A, CD127, IL-4R, GITR-L, TIM-4, CD153 (CD30L) , CD48, CD160, CD200R (OX2R) , and CD44. A “receptor-binding fragment” of a co-stimulatory ligand refers to a fragment of the ligand that retains its capacity to bind its receptor.

The term “treat” and its grammatical equivalents as used herein in connection with a disease or a condition, or a subject having a disease or a condition refer to an action that suppresses, eliminates, reduces, and/or ameliorates a symptom, the severity of the symptom, and/or the frequency of the symptom associated with the disease or disorder being treated. For example, when used in reference to a cancer or tumor, the term “treat” and its grammatical equivalents refer to an action that reduces the severity of the cancer or tumor, or retards or slows the progression of the cancer or tumor, including (a) inhibiting the growth, or arresting development of the cancer or tumor, (b) causing regression of the cancer or tumor, or (c) delaying, ameliorating or minimizing one or more symptoms associated with the presence of the cancer or tumor.

The term “administer” and its grammatical equivalents as used herein refer to the act of delivering, or causing to be delivered, a therapeutic or a pharmaceutical composition to the body of a subject by a method described herein or otherwise known in the art. The therapeutic can be a compound, a polypeptide, an antibody, a cell, or a population of cells. Administering a therapeutic or a pharmaceutical composition includes prescribing a therapeutic or a pharmaceutical composition to be delivered into the body of a subject. Exemplary forms of administration include oral dosage forms, such as tablets, capsules, syrups, suspensions; injectable dosage forms, such as intravenous (IV) , intramuscular (IM) , or intraperitoneal (IP) ; transdermal dosage forms, including creams, jellies, powders, or patches; buccal dosage forms; inhalation powders, sprays, suspensions, and rectal suppositories.

The terms “effective amount, ” “therapeutically effective amount, ” and their grammatical equivalents as used herein refer to the administration of an agent to a subject, either alone or as a part of a pharmaceutical composition and either in a single dose or as part of a series of doses, in an amount that is capable of having any detectable, positive effect on any symptom, aspect, or characteristics of a disease, disorder or condition when administered to the subject. The therapeutically effective amount can be ascertained by measuring relevant physiological effects. The exact amount required vary from subject to subject, depending on the age, weight, and general condition of the subject, the severity of the condition being treated, the judgment of the clinician, and the like. An appropriate “effective amount” in any individual case can be determined by one of ordinary skill in the art using routine experimentation.

The term “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” refers to a material that is suitable for drug administration to an individual along with an active agent without causing undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition.

The term “subject” as used herein refers to any animal (e.g., a mammal) , including, but not limited to, humans, non-human primates, canines, felines, rodents, and the like, which is to be the recipient of a particular treatment. A subject can be a human. A subject can have a particular disease or condition.

The term “autologous” as used herein refers to any material derived from the same individual to which it is later to be re-introduced into the individual.

The term “allogeneic” as used herein refers to a graft derived from a different animal of the same species.

Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

Exemplary genes and polypeptides are described herein with reference to GenBank numbers, GI numbers and/or SEQ ID NOs. It is understood that one skilled in the art can readily identify homologous sequences by reference to sequence sources, including but not limited to GenBank (ncbi. nlm. nih. gov/genbank/) and EMBL (embl. org/) .
5.2 Anti-HER2 antibodies and antigen-binding fragments

Provided herein are antibodies or antigen-binding fragments thereof that specifically bind HER2 (e.g., human HER2) . In some embodiments, provided herein are anti-HER2 antibodies. In some embodiments, the antibody is an IgA, IgD, IgE, IgG, or IgM antibody. In some embodiments, the antibody is an IgA antibody. In some embodiments, the antibody is an IgD antibody. In some embodiments, the antibody is an IgE antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgM antibody. In some embodiments, the antibodies provided herein can be an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody. In some embodiments, the antibody is an IgG1 antibody. In some embodiments, the antibody is an IgG2 antibody. In some embodiments, the antibody is an IgG3 antibody. In some embodiments, the antibody is an IgG4 antibody.

In some embodiments, provided herein are antigen-binding fragments of an anti-HER2 antibody. In some embodiments, antigen-binding fragments provided herein can be a single domain antibody (sdAb) , a heavy chain antibody (HCAb) , a Fab, a Fab’ , a F (ab’ ) 2, a Fv, a single-chain variable fragment (scFv) , or a (scFv) 2. In some embodiments, the antigen-binding fragment of an anti-HER2 antibody is a single domain antibody (sdAb) . In some embodiments, the antigen-binding fragment of an anti-HER2 antibody is a heavy chain antibody (HCAb) . In some embodiments, the antigen-binding fragment of an anti-HER2 antibody is a Fab. In some embodiments, the antigen-binding fragment of an anti-HER2 antibody is a Fab’ . In some embodiments, the antigen-binding fragment of an anti-HER2 antibody is a F (ab’ ) 2. In some embodiments, the antigen-binding fragment of an anti-HER2 antibody is a Fv. In some embodiments, the antigen-binding fragment of an anti-HER2 antibody is a scFv. In some embodiments, the antigen-binding fragment of an anti-HER2 antibody is a disulfide-linked scFv [ (scFv) 2] . In some embodiments, the antigen-binding fragment of an anti-HER2 antibody is a diabody (dAb) .

In some embodiments, the anti-HER2 antibodies or antigen-binding fragments provided herein comprise recombinant antibodies or antigen-binding fragments. In some embodiments, the anti-HER2 antibodies or antigen-binding fragments provided herein comprise monoclonal antibodies or antigen-binding fragments. In some embodiments, the anti-HER2 antibodies or antigen-binding fragments provided herein comprise polyclonal antibodies or antigen-binding fragments. In some embodiments, the anti-HER2 antibodies or antigen-binding fragments provided herein comprise camelid (e.g., camels, dromedary and llamas) antibodies or antigen-binding fragments. In some embodiments, the anti-HER2 antibodies or antigen-binding fragments provided herein comprise chimeric antibodies or antigen-binding fragments. In some embodiments, the anti-HER2 antibodies or antigen-binding fragments provided herein comprise humanized antibodies or antigen-binding fragments. In some embodiments, the anti-HER2 antibodies or antigen-binding fragments provided herein comprise human antibodies or antigen-binding fragments. In some embodiments, provided herein are anti-HER2 human scFvs.

In some embodiments, the anti-HER2 antibodies or antigen-binding fragments provided herein are isolated. In some embodiments, the anti-HER2 antibodies or antigen-binding fragments provided herein are substantially pure.

In some embodiments, the anti-HER2 antibody or antigen-binding fragment provided herein comprises a multispecific antibody or antigen-binding fragment. In some embodiments, the anti-HER2 antibody or antigen-binding fragment provided herein comprises a bispecific antibody or antigen-binding fragment. In some embodiments, provided herein is a Bi-specific T-cell engager (BiTE) . BiTEs are bispecific antibodies that bind to a T cell antigen (e.g., CD3) and a tumor antigen. BiTEs have been shown to induce directed lysis of target tumor cells and thus provide great potential therapies for cancers and other disorders. In some embodiments, provided herein are BiTEs that specifically bind CD3 and HER2. In some embodiments, the BiTEs comprises an anti-HER2 antibody or antigen-binding fragment provided herein. In some embodiments, the BiTEs comprises an anti-HER2 scFv provided herein.

In some embodiments, the anti-HER2 antibody or antigen-binding fragment provided herein comprises a monovalent antigen-binding site. In some embodiments, an anti-HER2 antibody or antigen-binding fragment comprises a monospecific binding site. In some embodiments, an anti-HER2 antibody or antigen-binding fragment comprises a bivalent binding site.

In some embodiments, an anti-HER2 antibody or antigen-binding fragment is a monoclonal antibody or antigen-binding fragment. Monoclonal antibodies can be prepared by any method known to those of skill in the art. One exemplary approach is screening protein expression libraries, e.g., phage or ribosome display libraries. Phage display is described, for example, in Ladner et al., U.S. Patent No. 5,223,409; Smith (1985) Science 228: 1315-1317; and WO 92/18619. In some embodiments, recombinant monoclonal antibodies are isolated from phage display libraries expressing variable regions or CDRs of a desired species. Screening of phage libraries can be accomplished by various techniques known in the art.

In some embodiments, monoclonal antibodies are prepared using hybridoma methods known to one of skill in the art. For example, using a hybridoma method, a mouse, rat, rabbit, hamster, or other appropriate host animal, is immunized as described above. In some embodiments, lymphocytes are immunized in vitro. In some embodiments, the immunizing antigen is a human protein or a fragment thereof. In some embodiments, the immunizing antigen is a human protein or a fragment thereof.

Following immunization, lymphocytes are isolated and fused with a suitable myeloma cell line using, for example, polyethylene glycol. The hybridoma cells are selected using specialized media as known in the art and unfused lymphocytes and myeloma cells do not survive the selection process. Hybridomas that produce monoclonal antibodies directed to a chosen antigen can be identified by a variety of methods including, but not limited to, immunoprecipitation, immunoblotting, and in vitro binding assays (e.g., flow cytometry, FACS, ELISA, SPR (e.g., Biacore) , and radioimmunoassay) . Once hybridoma cells that produce antibodies of the desired specificity, affinity, and/or activity are identified, the clones may be subcloned by limiting dilution or other techniques. The hybridomas can be propagated either in in vitro culture using standard methods or in vivo as ascites tumors in an animal. The monoclonal antibodies can be purified from the culture medium or ascites fluid according to standard methods in the art including, but not limited to, affinity chromatography, ion-exchange chromatography, gel electrophoresis, and dialysis.

In some embodiments, monoclonal antibodies are made using recombinant DNA techniques as known to one skilled in the art. For example, the polynucleotides encoding an antibody are isolated from mature B-cells or hybridoma cells, such as by RT-PCR using oligonucleotide primers that specifically amplify the genes encoding the heavy and light chains of the antibody, and their sequence is determined using standard techniques. The isolated polynucleotides encoding the heavy and light chains are then cloned into suitable expression vectors which produce the monoclonal antibodies when transfected into host cells such as E. coli, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin proteins.

In some embodiments, a monoclonal antibody is modified by using recombinant DNA technology to generate alternative antibodies. In some embodiments, the constant domains of the light chain and heavy chain of a mouse monoclonal antibody are replaced with the constant regions of a human antibody to generate a chimeric antibody. In some embodiments, the constant regions are truncated or removed to generate a desired antibody fragment of a monoclonal antibody. In some embodiments, site-directed or high-density mutagenesis of the variable region (s) is used to optimize specificity and/or affinity of a monoclonal antibody.

In some embodiments, an anti-HER2 antibody or antigen-binding fragment is a humanized antibody or antigen-binding fragment. Various methods for generating humanized antibodies are known in the art. Methods are known in the art for achieving high affinity binding with humanized antibodies. A non-limiting example of such a method is hypermutation of the variable region and selection of the cells expressing such high affinity antibodies (affinity maturation) . In addition to the use of display libraries, the specified antigen (e.g., recombinant HER2 or an epitope thereof) can be used to immunize a non-human animal, e.g., a rodent. In certain embodiments, rodent antigen-binding fragments (e.g., mouse antigen-binding fragments) can be generated and isolated using methods known in the art and/or disclosed herein. In some embodiments, a mouse can be immunized with an antigen (e.g., recombinant HER2 or an epitope thereof) .

In some embodiments, an anti-HER2 antibody or antigen-binding fragment is a human antibody or antigen-binding fragment. Human antibodies can be prepared using various techniques known in the art. In some embodiments, human antibodies are generated from immortalized human B lymphocytes immunized in vitro. In some embodiments, human antibodies are generated from lymphocytes isolated from an immunized individual. In any case, cells that produce an antibody directed against a target antigen can be generated and isolated. In some embodiments, a human antibody is selected from a phage library, where that phage library expresses human antibodies. Alternatively, phage display technology can be used to produce human antibodies and antibody fragments in vitro, from immunoglobulin variable region gene repertoires from unimmunized donors. Techniques for the generation and use of antibody phage libraries are well-known in the art. Once antibodies are identified, affinity maturation strategies known in the art, including but not limited to, chain shuffling and site-directed mutagenesis, can be employed to generate higher affinity human antibodies. In some embodiments, human antibodies are produced in transgenic mice that contain human immunoglobulin loci. Upon immunization these mice are capable of producing the full repertoire of human antibodies in the absence of endogenous immunoglobulin production.

The specific CDR sequences defined herein are generally based on a combination of Kabat definition. However, it is understood that reference to a heavy chain CDR or CDRs and/or a light chain CDR or CDRs of a specific antibody encompass all CDR definitions as known to those of skill in the art.

Anti-HER2 antibodies or antigen-binding fragments provided herein include the H13 clone, and the sequence features are described below.

In some embodiments, anti-HER2 antibodies or antigen-binding fragments provided herein (e.g., human HER2) comprise one, two, three, four, five, and/or six CDRs of any one of the antibodies described herein. In some embodiments, anti-HER2 antibodies or antigen-binding fragments provided herein comprise a VL comprising one, two, and/or three, LCDRs from Table 1. In some embodiments, anti-HER2 antibodies or antigen-binding fragments provided herein comprise a VH comprising one, two, and/or three HCDRs from Table 2. In some embodiments, anti-HER2 antibodies or antigen-binding fragments provided herein comprise one, two, and/or three LCDRs from Table 1 and one, two, and/or three HCDRs from Table 2.

Table 1 Amino acid sequences of light chain variable region CDRs (LCDRs) of anti-HER2

Table 2 Amino acid sequences of heavy chain variable region CDRs (HCDRs) of anti-HER2

In some embodiments, an anti-HER2 antibody or antigen-binding fragment thereof comprises a humanized antibody or antigen-binding fragment. In some embodiments, an anti-HER2 antibody or antigen-binding fragment thereof comprises a LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and/or HCDR3 from an antibody or antigen-binding fragment described herein. In some embodiments, an anti-HER2 antibody or antigen-binding fragment thereof comprises a variant of an anti-HER2 antibody or antigen-binding fragment described herein. In some embodiments, a variant of an anti-HER2 antibody or antigen-binding fragment comprises 1 to 30 amino acid substitutions, additions, and/or deletions in the anti-HER2 antibody or antigen-binding fragment. In some embodiments, a variant of an anti-HER2 antibody or antigen-binding fragment comprises one to 25 amino acid substitutions, additions, and/or deletions in the anti-HER2 antibody or antigen-binding fragment. In some embodiments, a variant of an anti-HER2 antibody or antigen-binding fragment comprises one to 20 substitutions, additions, and/or deletions in the anti-HER2 antibody or antigen-binding fragment. In some embodiments, a variant of an anti-HER2 antibody or antigen-binding fragment comprises 1 to 15 substitutions, additions, and/or deletions in the anti-HER2 antibody or antigen-binding fragment. In some embodiments, a variant of an anti-HER2 antibody or antigen-binding fragment comprises 1 to 10 substitutions, additions, and/or deletions in the anti-HER2 antibody or antigen-binding fragment. In some embodiments, a variant of an anti-HER2 antibody or antigen-binding fragment comprises one to five conservative amino acid substitutions, additions, and/or deletions in the anti-HER2 antibody or antigen-binding fragment. In some embodiments, a variant of an anti-HER2 antibody or antigen-binding fragment comprises one to three amino acid substitutions, additions, and/or deletions in the anti-HER2 antibody or antigen-binding fragment. In some embodiments, the amino acid substitutions, additions, and/or deletions are conservative amino acid substitutions. In some embodiments, the conservative amino acid substitution (s) is in a CDR of the antibody or antigen-binding fragment. In some embodiments, the conservative amino acid substitution (s) is not in a CDR of the antibody or antigen-binding fragment. In some embodiments, the conservative amino acid substitution (s) is in a framework region of the antibody or antigen-binding fragment.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind HER2 (e.g., human HER2) , comprising a light chain variable region (VL) comprising (1) a light chain CDR1 (LCDR1) having an amino acid sequence of SEQ ID NO: 1; (2) a light chain CDR2 (LCDR2) having an amino acid sequence consisting of SEQ ID NO: 2; or (3) a light chain CDR3 (LCDR3) having an amino acid sequence of SEQ ID NO: 3; or a variant thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and/or deletions in the LCDRs. In some embodiments, the variant has about 5 amino acid substitutions, additions, and/or deletions in the LCDRs.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind HER2 (e.g., human HER2) comprising a heavy chain variable region (VH) comprising (1) a heavy chain CDR1 (HCDR1) having an amino acid sequence of SEQ ID NO: 4; (2) a heavy chain CDR2 (HCDR2) having an amino acid sequence of SEQ ID NO: 5; or (3) a heavy chain CDR3 (HCDR3) having an amino acid sequence of SEQ ID NO: 6; or a variant thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and/or deletions in the HCDRs. In some embodiments, the variant has up about 5 amino acid substitutions, additions, and/or deletions in the HCDRs.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind HER2 (e.g., human HER2) having a VL and a VH. In some embodiments, the VL and VH are connected by a linker. The linker can be a flexible linker or a rigid linker. In some embodiments, the linker has the amino acid sequence of (GGGGS) n, n=1, 2, 3, 4, or 5 (SEQ ID NO: 21) . For example, the linker may have the amino acid sequence of GGGGS. In some embodiments, the linker has the amino acid sequence of (EAAAK) n, n=1, 2, 3, 4, or 5. For example, the linker may have the amino acid sequence of EAAAK (SEQ ID NO: 22) . In some embodiments, the linker has the amino acid sequence of (PA) nP, n=1, 2, 3, 4, or 5 (SEQ ID NO: 23) . In some embodiments, the linker has the amino acid sequence of GGGGSGGGGSGGGGS (SEQ ID NO: 24) .

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind HER2 (e.g., human HER2) having a VL and a VH, wherein the VL comprises LCDR1, CDR2 and CDR3 and the VH comprises HCDR1, CDR2 and CDR3, and wherein the LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3 have the amino acid sequences of SEQ ID NO: 1, 2, 3, 4, 5, and 6, respectively, or a variant thereof having up to about 5 amino acid substitutions, additions, and/or deletions in the CDRs.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind HER2 (e.g., human HER2) having a VL, comprising (1) a LCDR1 having the amino acid sequence of SEQ ID NO: 1, (2) a LCDR2 having the amino acid sequence of SEQ ID NO: 2, or (3) a LCDR3 having the amino acid sequence of SEQ ID NO: 3. The VL can have LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind HER2 having a VH, comprising (1) a HCDR1 having the amino acid sequence of SEQ ID NO: 4, (2) a HCDR2 having the amino acid sequence of SEQ ID NO: 5, or (3) a HCDR3 having the amino acid sequence of SEQ ID NO: 6. The VH can have HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind HER2 (e.g., human HER2) comprising (a) a VL that comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively; and (b) a VH that comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind HER2 (e.g., human HER2) comprising a VL having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence of SEQ ID NO: 7. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind HER2 (e.g., human HER2) comprising a VH having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence of SEQ ID NO: 8.

Table 3 Amino acid sequences of light chain variable regions (VL) and heavy chain variable region (VH) of anti-HER2 antibody

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind HER2 (e.g., human HER2) comprising: (a) a VL having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity sequence identity to an amino acid sequence of SEQ ID NO: 7; and (b) a VH having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity sequence identity to an amino acid sequence of SEQ ID NO: 8.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind HER2 (e.g., human HER2) comprising a VL and a VH, wherein the VL and VH have the amino acid sequences of SEQ ID NO: 7 and 8, respectively.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind HER2 (e.g., human HER2) comprising (a) a VL comprising LCDRs from a VL having an amino acid sequence of SEQ ID NO: 7; and/or (b) a VH comprising HCDRs from a VH having an amino acid sequence of SEQ ID NO: 8.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind HER2 (e.g., human HER2) comprising a VL and a VH, wherein the VL comprises LCDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 7, and the VH comprises HCDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 8.

In some embodiments, the anti-HER2 antibody or antigen-binding fragment thereof provided herein is the scFv designated as H13 (SEQ ID NO: 9) . In some embodiments, the anti-HER2 antibody or antigen-binding fragment thereof provided herein has an amino acid sequence that is at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%identical to SEQ ID NO: 9. In some embodiments, the anti-HER2 antibody or antigen-binding fragment thereof provided herein has a VL from HER2 (SEQ ID NO: 7) . In some embodiments, the anti-HER2 antibody or antigen-binding fragment thereof provided herein has a VH from H13 (SEQ ID NO: 8) . The anti-HER2 antibody or antigen-binding fragment thereof provided herein can have both a VL and a VH from H13. In some embodiments, the anti-HER2 antibody or antigen-binding fragment thereof provided herein has a VL that comprises LCDRs from the VL from H13 (SEQ ID NO: 7) . In some embodiments, the anti-HER2 antibody or antigen-binding fragment thereof provided herein has a VH that comprises HCDRs from the VH from H13 (SEQ ID NO: 8) . The anti-HER2 antibody or antigen-binding fragment thereof provided herein can have a VL comprising LCDRs and a VH comprising HCDRs from the VL and VH of H13, respectively. In some embodiments, the anti-HER2 antibody or antigen-binding fragment thereof provided herein is a variant of H13. The H13 variant can have a VL that is a variant of the VL of H13 having up to about 5 amino acid substitutions, additions, and/or deletions in SEQ ID NO: 7. The H13 variant can have a VH that is a variant of the VH of H13 having up to about 5 amino acid substitutions, additions, and/or deletions in SEQ ID NO: 8. The amino acid substitutions, additions, and/or deletions can be in the HCDRs or LCDRs. In some embodiments, the amino acid substitutions, additions, and/or deletions are not in the CDRs. In some embodiments, the variant of H13 has up to about 5 conservative amino acid substitutions. In some embodiments, the variant of H13 has up to 3 conservative amino acid substitutions.

In some embodiments, provided herein are also antibodies or antigen-binding fragments that compete with the antibody or antigen-binding fragment provided above for binding to HER2 (e.g., human HER2) . Antibodies that “compete with another antibody for binding to a target” refer to antibodies that inhibit (partially or completely) the binding of the other antibody to the target. Whether two antibodies compete with each other for binding to a target, i.e., whether and to what extent one antibody inhibits the binding of the other antibody to a target, can be determined using known competition experiments, e.g., surface plasmon resonance (SPR) analysis. In some embodiments, an anti-HER2 antibody or antigen-binding fragment competes with, and inhibits binding of another antibody or antigen-binding fragment to HER2 by at least 50%, 60%, 70%, 80%, 90%or 100%. Competition assays can be conducted as described, for example, in Ed Harlow and David Lane, Cold Spring Harb Protoc; 2006; doi: l0. H0l/pdb. prot4277 or in Chapter 11 of “Using Antibodies” by Ed Harlow and David Lane, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA 1999.

In some embodiments, provided herein are antibodies or antigen-binding fragments that compete with H13 for binding to HER2.

The present disclosure further contemplates additional variants and equivalents that are substantially homologous to the recombinant, monoclonal, chimeric, humanized, and human antibodies, or antibody fragments thereof, described herein. In some embodiments, it is desirable to improve the binding affinity of the antibody. In some embodiments, it is desirable to modulate biological properties of the antibody, including but not limited to, specificity, thermostability, expression level, effector function (s) , glycosylation, immunogenicity, and/or solubility. Those skilled in the art will appreciate that amino acid changes may alter post-translational processes of an antibody, such as changing the number or position of glycosylation sites or altering membrane anchoring characteristics.

Variations can be a substitution, deletion, or insertion of one or more nucleotides encoding the antibody or polypeptide that results in a change in the amino acid sequence as compared with the native antibody or polypeptide sequence. In some embodiments, amino acid substitutions are the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine, e.g., conservative amino acid replacements. Insertions or deletions can be in the range of about 1 to 5 amino acids. In some embodiments, the substitution, deletion, or insertion includes less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions relative to the parent molecule. In some embodiments, variations in the amino acid sequence that are biologically useful and/or relevant can be determined by systematically making insertions, deletions, or substitutions in the sequence and testing the resulting variant proteins for activity as compared to the parent protein.

It is known in the art that the constant region (s) of an antibody mediates several effector functions and these effector functions can vary depending on the isotype of the antibody. For example, binding of the C1 component of complement to the Fc region of IgG or IgM antibodies (bound to antigen) activates the complement system. Activation of complement is important in the opsonization and lysis of cell pathogens. The activation of complement also stimulates the inflammatory response and can be involved in autoimmune hypersensitivity. In addition, the Fc region of an antibody can bind a cell expressing a Fc receptor (FcR) . There are a number of Fc receptors which are specific for different classes of antibody, including IgG (gamma receptors) , IgE (epsilon receptors) , IgA (alpha receptors) and IgM (mu receptors) . Binding of antibody to Fc receptors on cell surfaces triggers a number of important and diverse biological responses including engulfment and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (called antibody-dependent cell cytotoxicity or ADCC) , release of inflammatory mediators, placental transfer, and control of immunoglobulin production. In some embodiments, anti-HER2 antibody or antigen-binding fragment described herein comprise at least one constant region of a human IgA antibody. In some embodiments, anti-HER2 antibody or antigen-binding fragment described herein comprise at least one constant region of a human IgD antibody. In some embodiments, anti-HER2 antibody or antigen-binding fragment described herein comprise at least one constant region of a human IgE antibody. In some embodiments, anti-HER2 antibody or antigen-binding fragment described herein comprise at least one constant region of a human IgG antibody. In some embodiments, anti-HER2 antibody or antigen-binding fragment described herein comprise at least one constant region of a human IgM antibody. In some embodiments, anti-HER2 antibody or antigen-binding fragment described herein comprise at least one constant region of a human IgG1 antibody. In some embodiments, anti-HER2 antibody or antigen-binding fragment described herein comprise at least one constant region of a human IgG2 antibody. In some embodiments, anti-HER2 antibody or antigen-binding fragment described herein comprise at least one constant region of a human IgG3 antibody. In some embodiments, anti-HER2 antibody or antigen-binding fragment described herein comprise at least one constant region of a human IgG4 antibody.

In some embodiments, at least one or more of the constant regions has been modified or deleted in the anti-HER2 antibody or antigen-binding fragment described herein. In some embodiments, the antibodies comprise modifications to one or more of the three heavy chain constant regions (CH1, CH2 or CH3) and/or to the light chain constant region (CL) . In some embodiments, the heavy chain constant region of the modified antibodies comprises at least one human constant region. In some embodiments, the heavy chain constant region of the modified antibodies comprises more than one human constant region. In some embodiments, modifications to the constant region comprise additions, deletions, or substitutions of one or more amino acids in one or more regions. In some embodiments, one or more regions are partially or entirely deleted from the constant regions of the modified antibodies. In some embodiments, the entire CH2 domain has been removed from an antibody (ΔCH2 constructs) . In some embodiments, a deleted constant region is replaced by a short amino acid spacer that provides some of the molecular flexibility typically imparted by the absent constant region. In some embodiments, a modified antibody comprises a CH3 domain directly fused to the hinge region of the antibody. In some embodiments, a modified antibody comprises a peptide spacer inserted between the hinge region and modified CH2 and/or CH3 domains.

In some embodiments, an anti-HER2 antibody or antigen-binding fragment comprises a Fc region. In some embodiments, the Fc region is fused via a hinge. The hinge can be an IgG1 hinge, an IgG2 hinge, or an IgG3 hinge. The amino acid sequences of the Fc region of human IgG1, IgG2, IgG3, and IgG4 are known to those of ordinary skill in the art. In some cases, Fc regions with amino acid variations have been identified in native antibodies. In some embodiments, the modified antibodies (e.g., modified Fc region) provide for altered effector functions that, in turn, affect the biological profile of the antibody. For example, in some embodiments, the deletion or inactivation (through point mutations or other means) of a constant region reduces Fc receptor binding of the modified antibody as it circulates. In some embodiments, the constant region modifications reduce the immunogenicity of the antibody. In some embodiments, the constant region modifications increase the serum half-life of the antibody. In some embodiments, the constant region modifications reduce the serum half-life of the antibody. In some embodiments, the constant region modifications decrease or remove ADCC and/or complement dependent cytotoxicity (CDC) of the antibody. In some embodiments, specific amino acid substitutions in a human IgG1 Fc region with corresponding IgG2 or IgG4 residues reduce effector functions (e.g., ADCC and CDC) in the modified antibody. In some embodiments, an antibody does not have one or more effector functions (e.g., “effectorless” antibodies) . In some embodiments, the antibody has no ADCC activity and/or no CDC activity. In some embodiments, the antibody does not bind an Fc receptor and/or complement factors. In some embodiments, the antibody has no effector function (s) . In some embodiments, the constant region modifications increase or enhance ADCC and/or CDC of the antibody. In some embodiments, the constant region is modified to eliminate disulfide linkages or oligosaccharide moieties. In some embodiments, the constant region is modified to add/substitute one or more amino acids to provide one or more cytotoxin, oligosaccharide, or carbohydrate attachment sites. In some embodiments, an anti-HER2 antibody or antigen-binding fragment comprises a variant Fc region that is engineered with substitutions at specific amino acid positions as compared to a native Fc region. In some embodiments, an anti-HER2 antibody or antigen-binding fragment described herein comprises an IgG1 heavy chain constant region that comprises one or more amino acid substitutions selected from the group consisting of K214R, L234A, L235E, G237A, D356E, and L358M, per EU numbering. In some embodiments, the IgG1 heavy chain constant region comprises one or more amino acid substitutions selected from the group consisting of K214R, L234A, L235E, G237A, A330S, P331S, D356E, and L358M, per EU numbering. In some embodiments, the IgG1 heavy chain constant region comprises one or more amino acid substitutions selected from the group consisting of K214R, C226S, C229S, and P238S, per EU numbering. In some embodiments, the IgG1 heavy chain constant region comprises one or more amino acid substitutions selected from the group consisting of K214R, D356E, and L358M, per EU numbering. In some embodiments, the IgG1 heavy chain constant region comprises one or more amino acid substitutions selected from the group consisting of S131C, K133R, G137E, G138S, Q196K, I199T, N203D, K214R, C226S, C229S, and P238S, per EU numbering.

In some embodiments, variants can include addition of amino acid residues at the amino-and/or carboxyl-terminal end of the antibody or polypeptide. The length of additional amino acids residues can range from one residue to a hundred or more residues. In some embodiments, a variant comprises an N-terminal methionyl residue. In some embodiments, the variant comprises an additional polypeptide/protein (e.g., Fc region) to create a fusion protein. In some embodiments, a variant is engineered to be detectable and may comprise a detectable label and/or protein (e.g., a fluorescent tag or an enzyme) .

The variant antibodies or antigen-binding fragments described herein can be generated using methods known in the art, including but not limited to, site-directed mutagenesis, alanine scanning mutagenesis, and PCR mutagenesis.

In some embodiments, a variant of an anti-HER2 antibody or antigen-binding fragment disclosed herein can retain the ability to bind HER2 to a similar extent, the same extent, or to a higher extent, as the parent antibody or antigen-binding fragment. In some embodiments, the variant can be at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%or more identical in amino acid sequence to the parent antibody or antigen-binding fragment. In certain embodiments, a variant of an anti-HER2 antibody or antigen-binding fragment comprises the amino acid sequence of the parent anti-HER2 antibody or antigen-binding fragment with one or more conservative amino acid substitution. Conservative amino acid substitutions are known in the art and include amino acid substitutions in which one amino acid having certain physical and/or chemical properties is exchanged for another amino acid that has the same or similar chemical or physical properties.

In some embodiments, a variant of an anti-HER2 antibody or antigen-binding fragment comprises the amino acid sequence of the parent antibody or antigen-binding fragment with one or more non-conservative amino acid substitutions. In some embodiments, a variant of an anti-HER2 antibody or antigen-binding fragment comprises the amino acid sequence of the parent binding antibody or antigen-binding fragment with one or more non-conservative amino acid substitution, wherein the one or more non-conservative amino acid substitutions do not interfere with or inhibit one or more biological activities of the variant (e.g., HER2 binding) . In certain embodiments, the one or more conservative amino acid substitutions and/or the one or more non-conservative amino acid substitutions can enhance a biological activity of the variant, such that the biological activity of the functional variant is increased as compared to the parent binding moiety.

In some embodiments, the variant can have 1, 2, 3, 4, or 5 amino acid substitutions in the CDRs (e.g., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3) of the binding moiety.

In some embodiments, anti-HER2 antibodies or antigen-binding fragments described herein are chemically modified naturally or by intervention. In some embodiments, the anti-HER2 antibodies or antigen-binding fragments have been chemically modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, and/or linkage to a cellular ligand or other protein. Any of numerous chemical modifications can be carried out by known techniques. The anti-HER2 antibodies or antigen-binding fragments can comprise one or more analogs of an amino acid (including, for example, unnatural amino acids) , as well as other modifications known in the art.

In some embodiments, an anti-HER2 antibody or antigen-binding fragment (e.g., an antibody) binds HER2 (e.g., human HER2) with a dissociation constant (KD) of about 1 μM or less, about 100 nM or less, about 40 nM or less, about 20 nM or less, about 10 nM or less, about 1 nM or less, about 0.1 nM or less, 50 pM or less, 10 pM or less, or 1 pM or less. In some embodiments, an anti-HER2 antibody or antigen-binding fragment binds HER2 (e.g., human HER2) with a KD of about 20 nM or less. In some embodiments, an anti-HER2 antibody or antigen-binding fragment binds HER2 (e.g., human HER2) with a KD of about 10 nM or less. In some embodiments, an anti-HER2 antibody or antigen-binding fragment binds HER2 (e.g., human HER2) with a KD of about 1 nM or less. In some embodiments, an anti-HER2 antibody or antigen-binding fragment binds HER2 (e.g., human HER2) with a KD of about 0.5 nM or less. In some embodiments, an anti-HER2 antibody or antigen-binding fragment binds HER2 (e.g., human HER2) with a KD of about 0.1 nM or less. In some embodiments, an anti-HER2 antibody or antigen-binding fragment binds HER2 (e.g., human HER2) with a KD of about 50 pM or less. In some embodiments, an anti-HER2 antibody or antigen-binding fragment binds HER2 (e.g., human HER2) with a KD of about 25 pM or less. In some embodiments, an anti-HER2 antibody or antigen-binding fragment binds HER2 (e.g., human HER2) with a KD of about 10 pM or less. In some embodiments, an anti-HER2 antibody or antigen-binding fragment binds HER2 (e.g., human HER2) with a KD of about 1 pM or less. In some embodiments, the dissociation constant of the binding agent (e.g., an antibody) for HER2 is the dissociation constant determined using a HER2 protein immobilized on a Biacore chip and the binding agent flowed over the chip. In some embodiments, the dissociation constant of the binding agent (e.g., an antibody) for HER2 is the dissociation constant determined using the binding agent captured by an anti-human IgG antibody on a Biacore chip and soluble HER2 flowed over the chip.

The anti-HER2 antibodies or antigen-binding fragments of the present disclosure can be analyzed for their physical, chemical and/or biological properties by various methods known in the art. In some embodiments, an anti-HER2 antibody is tested for its ability to bind HER2 (e.g., human HER2) . Binding assays include, but are not limited to, SPR (e.g., Biacore) , ELISA, and FACS. In addition, antibodies can be evaluated for solubility, stability, thermostability, viscosity, expression levels, expression quality, and/or purification efficiency.

Epitope mapping is a method of identifying the binding site, region, or epitope on a target protein where an antibody binds. A variety of methods are known in the art for mapping epitopes on target proteins. These methods include mutagenesis, including but not limited to, shotgun mutagenesis, site-directed mutagenesis, and alanine scanning; domain or fragment scanning; peptide scanning (e.g., Pepscan technology) ; display methods (e.g., phage display, microbial display, and ribosome/mRNA display) ; methods involving proteolysis and mass spectroscopy; and structural determination (e.g., X-ray crystallography and NMR) . In some embodiments, anti-HER2 antibodies or antigen-binding fragments described herein are characterized by assays including, but not limited to, N-terminal sequencing, amino acid analysis, HPLC, mass spectrometry, ion exchange chromatography, and papain digestion.

In some embodiments, an anti-HER2 antibody or antigen-binding fragment is conjugated to a cytotoxic agent or moiety. In some embodiments, an anti-HER2 antibody or antigen-binding fragment is conjugated to a cytotoxic agent to form an ADC (antibody-drug conjugate) . In some embodiments, the cytotoxic moiety is a chemotherapeutic agent including, but not limited to, methotrexate, adriamycin/doxorubicin, melphalan, mitomycin C, chlorambucil, duocarmycin, daunorubicin, pyrrolobenzodiazepines (PBDs) , or other intercalating agents. In some embodiments, the cytotoxic moiety is a microtubule inhibitor including, but not limited to, auristatins, maytansinoids (e.g., DM1 and DM4) , and tubulysins. In some embodiments, the cytotoxic moiety is an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof, including, but not limited to, diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S) , Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. In some embodiments, an antibody is conjugated to one or more small molecule toxins, such as calicheamicins, maytansinoids, trichothenes, and CC1065.

In some embodiments, an anti-HER2 antibody or antigen-binding fragment described herein is conjugated to a detectable substance or molecule that allows the agent to be used for diagnosis and/or detection. A detectable substance can include, but is not limited to, enzymes, such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase, and acetylcholinesterase; prosthetic groups, such as biotin and flavine (s) ; fluorescent materials, such as, umbelliferone, fluorescein, fluorescein isothiocyanate (FITC) , rhodamine, tetramethylrhodamine isothiocyanate (TRITC) , dichlorotriazinylamine fluorescein, dansyl chloride, cyanine (Cy3) , and phycoerythrin; bioluminescent materials, such as luciferase; radioactive materials, such as 212Bi, 14C, 57Co, 51Cr, 67Cu, 18F, 68Ga, 67Ga, 153Gd, 159Gd, 68Ge, 3H, 166Ho, 131I, 125I, 123I, 121I, 115In, 113In, 112In, 111In, 140La, 177Lu, 54Mn, 99Mo, 32P, 103Pd, 149Pm, 142Pr, 186Re, 188Re, 105Rh, 97Ru, 35S, 47Sc, 75Se, 153Sm, 113Sn, 117Sn, 85Sr, 99mTc, 201Ti, 133Xe, 90Y, 69Yb, 175Yb, 65Zn; positron emitting metals; and magnetic metal ions positron emitting metals; and magnetic metal ions.

An anti-HER2 antibody or antigen-binding fragment described herein can be attached to a solid support. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene. In some embodiments, an immobilized anti-HER2 antibody or antigen-binding fragment is used in an immunoassay. In some embodiments, an immobilized anti-HER2 antibody or antigen-binding fragment is used in purification of the target antigen (e.g., human HER2) .
5.3 CARs, TCRs and Genetically Engineered Immune Effector Cells

The anti-HER2 antibodies or antigen-binding fragments described herein can be used as part of a chimeric antigen receptor (CAR) or a T-Cell Receptor (TCR) that can be expressed in an immune effector cell for cancer treatment. As such, provided herein are also CARs and TCRs that specifically bind HER2, immune effector cells that express such CARs or TCRs, and the uses of such cells.
5.3.1 TCRs

Provided herein are T cell receptors (TCRs) that specifically bind HER2 ( “HER2 TCR” ) . TCRs are antigen-specific molecules that are responsible for recognizing antigenic peptides presented in the context of a product of the MHC on the surface of APCs or any nucleated cells. This system endows T cells, via their TCRs, with the potential ability to recognize the entire array of intracellular antigens expressed by a cell (including virus proteins) that are processed into short peptides, bound to an intracellular MHC molecule, and delivered to the surface as a peptide-MHC complex. This system allows foreign protein (e.g., mutated cancer antigen or virus protein) or aberrantly expressed protein to serve a target for T cells (e.g., Davis and Bjorkman (1988) Nature, 334, 395-402; Davis et al.(1998) Annu Rev Immunol, 16, 523-544) .

As such, in some embodiments, provided herein are TCRs comprising an anti-HER2 antibody or antigen-binding fragment described herein. The anti-HER2 antibody or antigen-binding fragment can be any anti-HER2 antibody or antigen-binding fragment described herein. For illustrative purposes, in some embodiments, the TCRs provided herein can comprise an anti-HER2 antibody or antigen-binding fragment having a VL and a VH, wherein the VL comprises LCDR1, CDR2 and CDR3 and the VH comprises HCDR1, CDR2 and CDR3, and wherein the LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3 have the amino acid sequences of SEQ ID NO: 1, 2, 3, 4, 5, and 6.

In some embodiments, the TCRs provided herein can comprise an anti-HER2 antibody or antigen-binding fragment that is the scFv designated as H13.
5.3.2 CARs

The term “chimeric antigen receptor” or “CAR” as used herein refers to an artificially constructed hybrid protein or polypeptide containing a binding moiety (e.g., an antibody) linked to immune cell (e.g., T cell) signaling or activation domains. In some embodiments, CARs are synthetic receptors that retarget T cells to tumor surface antigens (Sadelain et al., Nat. Rev. Cancer 3 (l) : 35-45 (2003) ; Sadelain et al., Cancer Discovery 3 (4) : 388-398 (2013) ) . CARs can provide both antigen binding and immune cell activation functions onto an immune cell such as a T cell. CARs have the ability to redirect T-cell specificity and reactivity toward a selected target in a non-MHC-restricted manner, exploiting the antigen-binding properties of monoclonal antibodies. The non-MHC-restricted antigen recognition can give T-cells expressing CARs the ability to recognize an antigen independent of antigen processing, thus bypassing a mechanism of tumor escape.

The typical structure of a CAR molecule includes an extracellular antigen-binding domain (e.g., scFv) , a transmembrane domain (TM) and an intracellular signaling domain. The extracellular antigen-binding domain of a CAR is usually derived from a monoclonal antibody (mAb) or from receptors or their ligands. Antigen binding by the CARs triggers phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) in the intracellular domain, initiating a signaling cascade required for cytolysis induction, cytokine secretion, and proliferation. CAR-expressing T cells (“CART” s) can be classified into three generations according to the presence of intracellular co-stimulatory signals.

In some embodiments, provided herein are CARs that specifically binds HER2 ( “HER2 CAR” ) . In some embodiments, the CAR can be a “first generation, ” “second generation” or “third generation” CAR (see, for example, Sadelain et al., Cancer Discov. 3 (4) : 388-398 (2013) ; Jensen et al., Immunol. Rev. 257: 127-133 (2014) ; Sharpe et al., Dis. Model Mech. 8 (4) : 337-350 (2015) ; June et al (2018) , Science 359 (6382) : 1361-1365) .

“First generation” CARs are typically composed of an extracellular antigen binding domain, for example, a single-chain variable fragment (scFv) , fused to a transmembrane domain, which is fused to a cytoplasmic/intracellular domain of the T cell receptor chain. “First generation” CARs typically have the intracellular domain from the CD3ζ-chain, which is the primary transmitter of signals from endogenous T cell receptors (TCRs) . “First generation” CARs can provide de novo antigen recognition and cause activation of both CD4+ and CD8+ T cells through their CD3ζ chain signaling domain in a single fusion molecule, independent of HLA-mediated antigen presentation. “Second-generation” CARs comprise a cancer antigen-binding domain fused to an intracellular signaling domain capable of activating immune effector cells such as T cells and a co-stimulatory domain designed to augment immune effector cell, such as T cell, potency and persistence (Sadelain et al., Cancer Discov. 3: 388-398 (2013) ) . CAR design can therefore combine antigen recognition with signal transduction, two functions that are physiologically borne by two separate complexes, the TCR heterodimer and the CD3 complex. “Second generation” CARs include an intracellular domain from various co-stimulatory receptors, for example, CD28, 4-1BB, ICOS, OX40, and the like, in the cytoplasmic tail of the CAR to provide additional signals to the cell. “Second generation” CARs provide both co-stimulation, for example, by CD28 or 4-1BB domains, and activation, for example, by a CD3ζ signaling domain. Studies have indicated that “Second Generation” CARs can improve the anti-tumor activity of T cells. In 2017, FDA approved two anti-CD19 CAR T cell products for the treatment of relapsed B-cell precursor acute lymphoblastic leukemia (B-ALL) and B-cell Non-Hodgkin Lymphoma. “Third generation” CARs provide multiple co-stimulation, for example, by comprising both CD28 and 4-1BB domains, and activation, for example, by comprising a CD3ζactivation domain.

As such, provided herein are CARs that specifically binds HER2, comprising, from N-terminus to C-terminus: (a) a HER2 binding domain comprising an anti-HER2 antibody or antigen-binding fragment provided herein, (b) a transmembrane domain, and (c) a cytoplasmic domain. The anti-HER2 antibody or antigen-binding fragment can be any anti-HER2 antibody or antigen-binding fragment described herein. For illustrative purposes, in some embodiments, the CARs provided herein can comprise an anti-HER2 antibody or antigen-binding fragment having a VL and a VH, wherein the VL comprises LCDR1, CDR2 and CDR3 and the VH comprises HCDR1, CDR2 and CDR3, and wherein the LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3 have the amino acid sequences of SEQ ID NO: 1, 2, 3, 4, 5, and 6, respectively.

In some embodiments, the CARs provided herein can comprise an anti-HER2 antibody or antigen-binding fragment that is the scFv designated as H13.

In some embodiments, the transmembrane domain of the CARs provided herein comprises a hydrophobic alpha helix that spans at least a portion of the membrane. Different transmembrane domains result in different receptor stability. After antigen recognition, receptors cluster and a signal is transmitted to the cell. In some embodiments, the transmembrane domain of the CAR provided herein can be derived from a protein or polypeptide that is naturally expressed in an immune effector cell. A transmembrane domain derived from a protein or polypeptide means that the transmembrane domain comprises the entire transmembrane region of the protein or polypeptide, or a fragment thereof. In some embodiments, the CAR provided herein can have a transmembrane domain derived from CD8, CD28, CD3ζ, CD4, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, BTLA, T-cell receptor (TCR) α chain, TCR β chain, or TCR ζ chain, CD3ε, CD45, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD154, or other polypeptides expressed in the immune effector cell. In some embodiments, the transmembrane domain of CARs provided herein comprises the transmembrane region of CD8, CD28, CD3ζ, CD4, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, BTLA, T-cell receptor (TCR) α chain, TCR β chain, or TCR ζ chain, CD3ε, CD45, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD154, or other polypeptides expressed in the immune effector cell.

In some embodiments, the transmembrane domain of CARs provided herein is derived from CD8. In some embodiments, the transmembrane domain comprises the transmembrane region of CD8. In some embodiments, the transmembrane domain is derived from CD28. In some embodiments, the transmembrane domain comprises the transmembrane region of CD28. In some embodiments, the transmembrane domain is derived from CD3ζ. In some embodiments, the transmembrane domain comprises the transmembrane region of CD3ζ.

In some embodiments, the transmembrane domain can be synthetic, in which case it comprises predominantly hydrophobic residues such as leucine and valine. Optionally, the transmembrane domain can be derived from a polypeptide that is not naturally expressed in the immune effector cell, so long as the transmembrane domain can function in transducing signal from antigen bound to the CAR to the intracellular signaling and/or co-stimulatory domains. In some embodiments, the transmembrane domain can comprise a triplet of phenylalanine, tryptophan and valine at each end. Optionally, a short oligo-or polypeptide linker, preferably between 2 and 10 amino acids in length can form the linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR. A glycine-serine doublet provides a particularly suitable linker.

Cytoplasmic domains of CARs provided herein can contain a signaling domain that functions in the immune effector cell expressing the CAR. Such a signaling domain can be, for example, derived from CD3ζ, Fc receptor γ, FcγRIIa, FcRβ (FcεR1b) , CD3γ, CD3δ, CD3ε, CD79a, CD79b, DAP10, or DAP12. A signaling domain can also be a combination of signaling domains derived from molecules selected from CD3ζ, Fc receptor γ, FcγRIIa, FcRβ (FcεR1b) , CD3γ, CD3δ, CD3ε, CD79a, CD79b, DAP10, and DAP12. A signaling domain derived from a protein or polypeptide refers to the domain of the protein or polypeptide that is responsible for activating the immune effector cell (e.g., a T cell) , or a fragment thereof that retains its activation function. In general, the signaling domain induces persistence, trafficking and/or effector functions in the transduced immune effector cells such as T cells (Sharpe et al., Dis. Model Mech. 8: 337-350 (2015) ; Finney et al., J. Immunol. 161: 2791-2797 (1998) ; Krause et al., J. Exp. Med. 188: 619-626 (1998) ) . The signaling domain of a protein or polypeptide can be the intracellular domain of the protein or polypeptide. In some embodiments, the signaling domain comprises the intracellular domain of CD3ζ, FcRγ, FcγRIIa, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, DAP10, DAP12, or any combination thereof.

In some embodiments, the cytoplasmic domain of CARs provided herein comprises a signaling domain derived from CD3ζ. In some embodiments, the signaling domain comprises the intracellular domain of CD3ζ.

In some embodiments, the cytoplasmic domain of CARs provided herein further comprises a co-stimulatory domain. In some embodiments, the cytoplasmic domain of CARs provided herein further comprises two co-stimulatory domains. Such a co-stimulatory domain can provide increased activation of an immune effector cell (e.g., T cell) . A co-stimulatory signaling domain can be derived from, for example, CD28, 4-1BB (CD137) , OX40, ICOS, DAP10, 2B4, CD27, CD30, CD40, CD2, CD7, LIGHT, TIGIT, GITR, TLR, DR3, or CD43. A co-stimulatory domain derived from a protein or polypeptide refers to the domain of the protein or polypeptide that is responsible for providing increased activation of an immune effector cell (e.g., T cell) , or a fragment thereof that retains its activation function. In some embodiments, the co-stimulatory domain of CARs provided herein comprises the intracellular domain of CD28, 4-1BB (CD137) , OX40, ICOS, DAP10, 2B4, CD27, CD30, CD40, CD2, CD7, LIGHT, TIGIT, GITR, TLR, DR3, or CD43.

In some embodiments, the cytoplasmic domain of CARs provided herein comprises a co-stimulatory domain derived from CD28. In some embodiments, the co-stimulatory domain comprises the intracellular domain of CD28. In some embodiments, the cytoplasmic domain comprises a co-stimulatory domain derived from 4-1BB. In some embodiments, the co-stimulatory domain comprises the intracellular domain of 4-1BB.

CARs comprising an intracellular domain that comprises a co-stimulatory domain derived from 4-1BB, ICOS or DAP-10 have been described previously (see U.S. 7,446,190, which is incorporated herein by reference, which also describes representative sequences for 4-1BB, ICOS and DAP-10) . In some embodiments, the cytoplasmic domain of a CAR can comprise two co-stimulatory domains derived from two co-stimulatory receptors, such as CD28 and 4-1BB (see Sadelain et al., Cancer Discov. 3 (4) : 388-398 (2013) ) , or CD28 and OX40, or other combinations of co-stimulatory ligands, as disclosed herein.

The extracellular domain of a CAR can be fused to a leader or a signal peptide that directs the nascent protein into the endoplasmic reticulum and subsequent translocation to the cell surface. It is understood that, once a polypeptide containing a signal peptide is expressed at the cell surface, the signal peptide has generally been proteolytically removed during processing of the polypeptide in the endoplasmic reticulum and translocation to the cell surface. Thus, a polypeptide such as a CAR is generally expressed at the cell surface as a mature protein lacking the signal peptide, whereas the precursor form of the polypeptide includes the signal peptide. A signal peptide or leader can be essential if a CAR is to be glycosylated and/or anchored in the cell membrane. The signal sequence or leader is a peptide sequence generally present at the N-terminus of newly synthesized proteins that directs their entry into the secretory pathway. The signal peptide is covalently joined to the N-terminus of the extracellular antigen-binding domain of a CAR as a fusion protein. Any suitable signal peptide, as are well known in the art, can be applied to a CAR to provide cell surface expression in an immune cell (see Gierasch Biochem. 28: 923-930 (1989) ; von Heijne, J. Mol. Biol. 184 (1) : 99–105 (1985) ) . Particularly useful signal peptides can be derived from cell surface proteins naturally expressed in the immune cell provided herein, including any of the signal peptides of the polypeptides disclosed herein. Thus, any suitable signal peptide can be utilized to direct a CAR to be expressed at the cell surface of an immune effector cell provided herein.

In some embodiments, a CAR can also comprise a spacer region or sequence that links the domains of the CAR to each other. For example, a spacer can be included between a signal peptide and an antigen binding domain, between the antigen binding domain and the transmembrane domain, between the transmembrane domain and the intracellular domain, and/or between domains within the intracellular domain, for example, between a stimulatory domain and a co-stimulatory domain. The spacer region can be flexible enough to allow interactions of various domains with other polypeptides, for example, to allow the antigen binding domain to have flexibility in orientation in order to facilitate antigen recognition. The spacer region can be, for example, the hinge region from an IgG, the CH2CH3 (constant) region of an immunoglobulin, and/or portions of CD3 (cluster of differentiation 3) or some other sequence suitable as a spacer. In some embodiments, a CAR disclosed herein comprises a hinge domain that connects the HER2 binding domain and the transmembrane domain. In some embodiments, the hinge domain comprises human CD8 hinge domain. In some embodiments, the hinge domain comprises human CD28 hinge domain.

Provided below are some exemplary molecules from which domains of the CARs provided herein can be derived.

CD3ζ. CD3ζ comprises 3 Immune-receptor-Tyrosine-based-Activation-Motifs (ITAMs) , and transmits an activation signal to the cell, for example, a cell of the lymphoid lineage such as a T cell, after antigen is bound. A CD3ζ polypeptide can have an amino acid sequence corresponding to the sequence having GenBank No. NP_932170 (NP_932170.1, GI: 37595565; see below) or fragments thereof. In some embodiments, a CD3ζ signaling domain has an amino acid sequence of amino acids 52 to 164 of the CD3ζ polypeptide sequence provided below, or a fragment thereof that is sufficient for signaling activity. See GenBank NP_932170 for reference to domains within CD3ζ, for example, signal peptide, amino acids 1 to 21; extracellular domain, amino acids 22 to 30; transmembrane region, amino acids 31 to 51; intracellular domain, amino acids 52 to 164. In some embodiments, a CAR can have a transmembrane domain derived from CD3ζ. The transmembrane domain can comprise the transmembrane region of CD3ζ (e.g., amino acids 31 to 51 of the sequence below) , or a fragment thereof. In some embodiments, the cytoplasmic domain of a CAR can comprise a signaling domain derived from CD3ζ. In some embodiments, a signaling domain of CD3ζ can comprise the intracellular domain of CD3ζ (e.g., amino acids 52 to 164 of the sequence below) , or a fragment thereof. It is understood that sequences of CD3ζ that are shorter or longer than a specific delineated domain can be included in a CAR, if desired.

4-1BB. 4-1BB, also referred to as tumor necrosis factor receptor superfamily member 9, can act as a tumor necrosis factor (TNF) ligand and have stimulatory activity. A 4-1BB polypeptide can have an amino acid sequence corresponding to the sequence having GenBank No. P41273 (P41273.1, GI: 728739) or NP_001552 (NP_001552.2, GI: 5730095) . In some embodiments, a CAR can comprise a transmembrane domain derived from 4-1BB.

CD8. Cluster of differentiation 8 (CD8) is a transmembrane glycoprotein that serves as a co-receptor for the T cell receptor (TCR) . CD8 binds to a major histocompatibility complex (MHC) molecule and is specific for the class I MHC protein. In some embodiments, a CAR can comprise a transmembrane domain derived from CD8. A CD8 polypeptide can have an amino acid sequence corresponding to the sequence having GenBank No. NP_001139345.1 (GI: 225007536) , or fragments thereof. See GenBank NP_001139345.1 for reference to domains within CD8, for example, signal peptide, amino acids 1 to 21; extracellular domain, amino acids 22 to 182; transmembrane domain amino acids, 183 to 203; intracellular domain, amino acids 204 to 235. In some embodiments, a CAR can comprise a hinge domain derived from CD8.

As such, for exemplary purposes, a CAR disclosed herein can comprise, from N-terminus to the C-terminus, an anti-HER2 antibody or antigen-binding fragment (e.g., scFvs disclosed herein) , a hinge (e.g., CD8 hinge) , a transmembrane region (e.g., CD8 transmembrane region) , a costimulatory domain (e.g., the intracellular domain of 4-1BB) , and a signaling domain (e.g., the T cell signaling domain of CD3ζ) .

In some embodiments, the CAR comprises the amino acid sequence as set forth in SEQ ID NO: 10.
5.4 LACOSTIM fusion proteins

In some embodiments, cells provided herein recombinantly express a fusion protein. In some embodiments, cells are genetically modified to express the fusion protein. In some embodiments, cells provided herein are genetically modified to express the fusion protein provided herein and the CAR provided herein.

In some embodiments, the fusion protein is a membrane protein. In some embodiments, the fusion protein is a soluble protein. In some embodiments, the fusion protein is a bispecific antibody.

In some embodiments, the fusion protein comprising a first domain that activates an antigen-presenting cell (APC) and a second domain that activates an immune effector cell, wherein (i) the first domain comprises (a) a ligand that binds an activation receptor of the APC, or a receptor-binding fragment thereof, or (b) an antibody that binds an activation receptor of the APC, or an antigen-binding fragment thereof; and (ii) the second domain comprises (a) a co-stimulatory receptor of the immune effector cell, or a functional fragment thereof, (b) a co-stimulatory ligand of the immune effector cell, or a receptor-binding fragment thereof, or (c) an antibody that binds a co-stimulatory receptor of the immune effector cell, or an antigen-binding fragment thereof.

In some embodiments, the APC is selected from the group consisting of a dendritic cell, a macrophage, a myeloid derived suppressor cell, a monocyte, a B cell, a T cell, and a Langerhans cell. In some embodiments, the activation receptor of the APC is selected from the group consisting of CD40, CD80, CD86, CD91, DEC-205 and DC-SIGN. In some embodiments, the first domain comprises the ligand that binds CD40, CD80, CD86, CD91, DEC-205 or DC-SIGN. In some embodiments, the first domain comprises the extracellular domain of CD40 Ligand (CD40L ECD) . In some embodiments, the first domain comprises an antibody that binds the activation receptor of the APC, or an antigen-binding fragment thereof. In some embodiments, the first domain is an anti-CD40 antibody or an antigen-binding fragment thereof. In some embodiments, the first domain is a monoclonal antibody. In some embodiments, the first domain is a chimeric, humanized, or human antibody. In some embodiments, the first domain is a Fab, Fab’ , F (ab’ ) 2, Fv, scFv, (scFv) 2, single chain antibody, dual variable region antibody, diabody, nanobody, or single variable region antibody. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof comprises a heavy chain variable domain and/or a light chain variable domain. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof is a scFv.

In some embodiments, the second domain of the fusion proteins provided herein is a co-stimulatory receptor of the immune effector cell, or a functional fragment thereof. In some embodiments, the second domain of the fusion proteins provided herein comprises a cytoplasmic domain of the co-stimulatory receptor. In some embodiments, the co-stimulatory receptor is selected from the group consisting of CD28, 4-1BB, ICOS, CD27, OX40, DAP10, 2B4, CD30, CD2, LIGHT, GITR, TLR, DR3, and CD43. In some embodiments, the co-stimulatory receptor is CD28 or 4-1BB. In some embodiments, the co-stimulatory receptor is CD28. In some embodiments, the co-stimulatory receptor is 4-1BB. In some embodiments, the second domain further comprises the transmembrane domain of the co-stimulatory receptor. In some embodiments, the second domain of fusion proteins provided herein comprises a functional fragment of CD28, which comprises a portion of an intracellular/cytoplasmic domain of CD28 that can function as a co-stimulatory signaling domain. In some embodiments, the second domain comprises a functional fragment of CD28. In some embodiments, the second domain comprises a CD28 extracellular domain, a CD28 transmembrane domain and a CD28 intracellular domain.

In some embodiments, the second domain is a co-stimulatory ligand of the immune effector cell, or a receptor-binding fragment thereof. In some embodiments, the co-stimulatory ligand is selected from the group consisting of CD58, HER2, CD83, CD80, CD86, CD137L, CD252, CD275, CD54, CD49a, CD112, CD150, CD155, CD265, CD270, TL1A, CD127, IL-4R, GITR-L, TIM-4, CD153, CD48, CD160, CD200R, and CD44. In some embodiments, the second domain is an antibody that binds the co-stimulatory receptor, or an antigen-binding fragment thereof. In some embodiments, the co-stimulatory receptor is selected from the group consisting of CD28, 4-1BB, ICOS, CD27, OX40, DAP10, 2B4, CD30, CD2, LIGHT, GITR, TLR, DR3, and CD43. In some embodiments, the second domain is a monoclonal antibody. In some embodiments, the second domain is a chimeric, humanized, or human antibody.

In some embodiments, the N-terminus of the first domain of the fusion protein is linked to the C-terminus of the second domain of the fusion protein. In some embodiments, the N-terminus of the second domain of the fusion protein is linked to the C-terminus of the first domain of the fusion protein. In some embodiments, the first domain and the second domain of the fusion protein are linked via a linker. The linker can be a flexible linker or a rigid linker. In some embodiments, the linker has the amino acid sequence of (GGGGS) n, n=1, 2, 3, 4, or 5 (SEQ ID NO: 21) . In some embodiments, the linker has the amino acid sequence of (EAAAK) n, n=1, 2, 3, 4, or 5 (SEQ ID NO: 22) . In some embodiments, the linker has the amino acid sequence of (PA) nP, n=1, 2, 3, 4, or 5 (SEQ ID NO: 23) . In some embodiments, the linker has the amino acid sequence of GGGGSGGGGSGGGGS (SEQ ID NO: 24) . In some embodiments, the linker is a CD8 hinge (SEQ ID NO: 13) . In some embodiments, the linker is a CD28 hinge (SEQ ID NO: 25) . In some embodiments, the linker is an IgG1 hinge (SEQ ID NO: 26) . In some embodiments, the linker can be a trimerization motif selected from the group consisting of a T4 fibritin trimerization motif (SEQ ID NO: 27) , an isoleucine zipper (SEQ ID NO: 28 or 29) , a GCN4II motif (SEQ ID NO: 30 or 31) , a Matrilin-1 motif (SEQ ID NO: 32 or 33) , and a collagen XV trimerization motif (SEQ ID NO: 34) .

In some embodiments, the first domain of the fusion protein comprises a CD40L ECD, and the second domain of the fusion protein comprises a CD28 cytoplasmic domain. In some embodiments, the first domain of the fusion protein comprises a CD40L. In some embodiments, the first domain of the fusion protein comprises a CD40L ECD and the second domain of the fusion protein comprises an anti-CD28 antibody or an antigen-binding fragment thereof. In some embodiments, the first domain of the fusion protein comprises an anti-CD40 antibody or an antigen-binding fragment thereof, and the second domain of the fusion protein comprises an anti-CD28 antibody or an antigen-binding fragment thereof. In some embodiments, the fusion protein is a bispecific antibody having two scFvs that bind to CD40 and CD28 respectively.

In some embodiments, the fusion protein further comprises a signal peptide domain. In some embodiments, the C-terminus of the signal peptide is linked to the N-terminus of the first domain. In some embodiments, the signal peptide comprises a signal peptide derived from CD8. In some embodiments, the signal peptide comprises a signal peptide derived from CD40L.

In some embodiments, cells provided herein recombinantly express a fusion protein comprising a first domain that activates an antigen-presenting cell (APC) and a second domain that activates an immune effector cell, wherein: (i) the first domain comprises an anti-CD40 antibody or antigen-binding fragment thereof; and (ii) the second domain comprises a CD28 transmembrane region and a CD28 cytoplasmic domain. In some embodiments, cells provided herein express CARs provided herein and express the fusion protein. Expression of the fusion proteins disclosed herein can not only promote the proliferation and activation of immune effector cells (e.g., T cells) , but also stimulate the maturation and epitope spreading activities of antigen-presenting cells. In some embodiments, expression of the fusion proteins in genetically engineered T cell disclosed herein helps them overcome immunosuppression in tumor microenvironment mediated by such as the PD1/PD-L1 signaling, regulatory T cells (Tregs) and TGF-beta signaling, and enhances their anti-tumor activities. The fusion proteins of the present disclosure are also referred to as Lymphocytes-Antigen presenting cells Co-stimulators ( “LACOSTIMs” , “LACOSTIM” , “LACO-Stims” , “LACO-Stim” or “LACO” ) . For example, the fusion protein described herein comprises Lymphocytes-Antigen presenting cells Co-stimulators described in US Patent No. 11667723, the disclosure of US Patent No. 11667723 is hereby incorporated by reference.

CD40: CD40 is a 48 kD transmembrane glycoprotein surface receptor that is a member of the Tumor Necrosis Factor Receptor superfamily (TNFRSF) . Exemplary amino acid sequences of human CD40 are described (see, e.g., Accession: ALQ33424.1, GenBank NP_001241.1, GI: 957949089) , and SEQ ID NO: 35 is the amino acid sequence of human CD40. CD40 was initially characterized as a co-stimulatory receptor expressed on APCs that played a central role in B and T cell activation. The ligand for CD40, CD154 (also known as TRAP, T-BAM, CD40 Ligand or CD40L) is a type II integral membrane protein. See GenBank NP_001241.1 for reference to domains within CD40, for example, signal peptide, amino acids 1 to 20; extracellular domain, amino acids 21 to 193; transmembrane domain, amino acids 194 to 215; intracellular domain, amino acids 216 to 277.

CD28: Cluster of Differentiation 28 (CD28) is a protein expressed on T cells that provides co-stimulatory signals for T cell activation and survival. CD28 is the receptor for CD80 (B7.1) and CD86 (B7.2) proteins. A CD28 polypeptide can have an amino acid sequence corresponding to the sequence having GenBank No. P10747 (P10747.1, GI: 115973) or NP_006130 (NP_006130.1, GI: 5453611) , the amino acid sequence of SEQ ID NO: 37, or fragments thereof. See GenBank NP_006130 for reference to domains within CD28, for example, signal peptide, amino acids 1 to 18; extracellular domain, amino acids 19 to 152; transmembrane domain, amino acids 153 to 179; intracellular domain, amino acids 180 to 220.

In some embodiments, the first domain comprises an anti-CD40 antibody or an antigen-binding fragment thereof. In some embodiments, the first domain comprises an anti-CD40 scFv. In some embodiments, the first domain comprises an anti-CD40 scFv having amino acid sequences of SEQ ID NOs: 38-49 as listed in table 4.

Table 4: CD40 scFvs

In some embodiments, the second domain is an antibody or an antigen-binding fragment that binds CD28. In some embodiments, the second domain comprises a CD28 cytoplasmic domain. In some embodiments, the second domain comprises a CD28 cytoplasmic domain having amino acid sequences of SEQ ID NO: 36. In some embodiments, the second domain comprises a CD28 scFv. In some embodiments, the second domain comprises a CD28 scFv having VH (SEQ ID NO: 62) and/or VL(SEQ ID NO: 63) . In some embodiments, the second domain comprises a CD28 scFv having an amino acid sequence of SEQ ID NO: 64.

Table 5: CD28 scFv

In some embodiments, the fusion protein is at least 85%, 90%, 95%, 98%, 99%or 100%identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 50-61 and 65-76. In some embodiments, the fusion protein comprises an amino acid sequence of SEQ ID NO: 55. In some embodiments, the fusion protein comprises an amino acid sequence of SEQ ID NO: 70. In some embodiments, the fusion protein comprises an amino acid sequence of SEQ ID NO: 61. In some embodiments, the fusion protein comprises an amino acid sequence of SEQ ID NO: 76.
5.5 Polynucleotides and Vectors

Also provided herein are polynucleotides that encode a polypeptide (e.g., an anti-HER2 antibody or antigen-binding fragment or a CAR that specifically binds HER2, or a fusion protein) described herein. The term “polynucleotides that encode a polypeptide” encompasses a polynucleotide which includes only coding sequences for the polypeptide as well as a polynucleotide which includes additional coding and/or non-coding sequences. The polynucleotides of the disclosure can be in the form of RNA or in the form of DNA. DNA can be cDNA, genomic DNA, or synthetic DNA, and can be double-stranded or single-stranded. Single-stranded DNA can be the coding strand or non-coding (anti-sense) strand. The polynucleotides of the disclosure can be mRNA.

Vectors and cells comprising the polynucleotides described herein are also provided. In some embodiments, provided herein are vectors comprising a polynucleotide provided herein. The vectors can be expression vectors. In some embodiments, vectors provided herein comprise a polynucleotide encoding an anti-HER2 antibody or antigen-binding fragment described herein. In some embodiments, vectors provided herein comprise a polynucleotide encoding a polypeptide that is part of an anti-HER2 antibody or antigen-binding fragment described herein. In some embodiments, vectors provided herein comprise a polynucleotide encoding a CAR or TCR described herein. In some embodiments, vectors provided herein comprise a polynucleotide encoding a polypeptide that is part of a CAR or TCR described herein. A wide variety of expression host/vector combinations can be employed. Useful expression vectors for eukaryotic hosts include, for example, vectors comprising expression control sequences from SV40, bovine papilloma virus, adenovirus, lentivirus, and cytomegalovirus. Useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from E. coli, including pCR1, pBR322, pMB9 and their derivatives, and wider host range plasmids, such as M13 and other filamentous single-stranded DNA phages.

The present disclosure also provides host cells comprising the polypeptides described herein, polynucleotides encoding polypeptides described herein, or vectors comprising such polynucleotides. In some embodiments, provided herein are host cells comprising a vector comprising a polynucleotide disclosed herein. In some embodiments, host cells provided herein comprise a vector comprising a polynucleotide encoding an anti-HER2 antibody or antigen-binding fragment described herein. In some embodiments, host cells provided herein comprise a vector comprising a polynucleotide encoding a polypeptide that is part of an anti-HER2 antibody or antigen-binding fragment described herein. In some embodiments, host cells provided herein comprise a polynucleotide encoding an anti-HER2 antibody or antigen-binding fragment described herein. In some embodiments, the cells produce the anti-HER2 antibodies or antigen-binding fragments described herein. In some embodiments, host cells provided herein comprise a vector comprising a polynucleotide encoding a CAR or TCR described herein. In some embodiments, host cells provided herein comprise a vector comprising a polynucleotide molecule encoding a polypeptide that is part of a CAR or TCR described herein. In some embodiments, host cells provided herein comprise a polynucleotide encoding a CAR or TCR described herein. In some embodiments, the host cells produce the HER2 CARs or TCR described herein.
5.6 Cells

Provided herein are cells comprising the polynucleotides disclosed herein. In some embodiments, provided herein are cells comprising a polynucleotide that encodes a polypeptide disclosed herein. In some embodiments, provided herein are cells comprising a vector having a polynucleotide disclosed herein. In some embodiments, provided herein are cells recombinantly expressing a polypeptide disclosed herein. The polypeptide can be an anti-HER2 antibody or antigen-binding fragment. The polypeptide can be HER2 CAR. The polypeptide can be a HER2 TCR. In some embodiments, the cells provided herein can further express the fusion proteins disclosed herein or comprise a polynucleotide encoding a fusion protein disclosed herein. The fusion protein can be any LACOSTIM molecules disclosed herein.

In some embodiments, the cells provided herein express a CAR disclosed herein and a fusion protein disclosed herein. In some embodiments, cells provided herein comprise a first polynucleotide encoding a fusion protein provided herein, and a second polynucleotide encoding a CAR. In some embodiments, the cells express a TCR disclosed herein and a fusion protein disclosed herein. In some embodiments, the cells comprise a first polynucleotide encoding a fusion protein provided herein, and a second polynucleotide encoding a TCR.

In some embodiments, the genetically engineered immune effector cells provided herein comprise a polynucleotide that comprises a first fragment encoding a CAR and a second fragment encoding a fusion protein. In some embodiments, the genetically engineered immune effector cells provided herein comprise a polynucleotide that comprises a first fragment encoding a TCR and a second fragment encoding a fusion protein. In some embodiments, the polynucleotide has the first fragment and the second fragment from N-terminus to the C-terminus. In some embodiments, the polynucleotide has the second fragment and the first fragment from N-terminus to the C-terminus. In some embodiments, the cells provided herein comprise a polynucleotide having a first fragment encoding a CAR or a TCR provided herein and a second fragment that encodes a fusion protein disclosed herein. The first fragment and the second fragment can be linked by a nucleotide sequence encoding a linker. The linker can be a self-cleaving linker. In some embodiments, the first and second fragment are linked by a nucleotide sequence encoding a 2A peptide. In some embodiments, the linker is an F2A peptide (e.g.: SEQ ID NO: 101) . In some embodiments, the 2A linker is a T2A peptide (e.g.: SEQ ID NO: 102) . In some embodiments, the linker is an Furin-GS2-T2A peptide (e.g.: SEQ ID NO: 103) . In some embodiments, the first fragment (CAR/TCR-encoding) is located at the 5’ end of the second fragment (fusion protein-encoding) . In some embodiments, the first fragment (CAR/TCR-encoding) is located at the 3’ end of the second fragment (fusion protein encoding) .

In some embodiments, cells co-expressing the first and second fragment have the amino acid sequences or the nucleoside sequences of SEQ ID NOs: 77-100, In some embodiments, the first and second fragment have the amino acid sequence listed in table 6.

Table 6: CAR AND LACOSTIM sequences in Co-expressing LACOSTIM CAR-T Cells:

In some embodiments, cells provided herein are immune effector cells. In some embodiments, the immune effector cells are selected from the group consisting of T cells, B cell, natural killer (NK) cells, NKT cells, macrophages, granulocytes, neutrophils, eosinophils, mast cells, and basophils. In some embodiments, the immune effector cells provided herein are selected from the group consisting of T cells, NK cells, NKT cells, macrophages, neutrophils, and granulocytes. In some embodiments, the immune effector cell provided herein is a T cell. In some embodiments, the immune effector cell provided herein is an NK cell. In some embodiments, the immune effector cell provided herein is an NKT cell. In some embodiments, the immune effector cell provided herein is a macrophage. In some embodiments, the immune effector cell provided herein is a neutrophil. In some embodiments, the immune effector cell provided herein is a granulocyte.

In some embodiments, the immune effector cells provided herein can be genetically engineered. In some embodiments, the genetically engineered immune effector cells provided herein are isolated. In some embodiments, the genetically engineered immune effector cells provided herein are substantially pure.

As such, in some embodiments, provided herein are immune effector cells recombinantly expressing a polypeptide (e.g., an antibody or a CAR) disclosed herein. Provided herein are also immune effector cells (e.g., T cells) comprising a polynucleotide encoding a polypeptide (e.g., an antibody or a CAR) disclosed herein, or a vector having a polynucleotide disclosed herein. In some embodiments, provided herein are immune effector cells (e.g., T cells) comprising a polynucleotide that encodes an anti-HER2 antibody or antigen-binding fragment disclosed herein. In some embodiments, provided herein are immune effector cells (e.g., T cells) recombinantly expressing an anti-HER2 antibody or antigen-binding fragment disclosed herein. In some embodiments, provided herein are immune effector cells comprising a polynucleotide that encodes a HER2 CAR disclosed herein. In some embodiments, provided herein are immune effector cells (e.g., T cells) recombinantly expressing a HER2 CAR disclosed herein (e.g., HER2 CART cell) . In some embodiments, provided herein are immune effector cells comprising a polynucleotide that encodes a HER2 TCR disclosed herein. In some embodiments, provided herein are immune effector cells (e.g., T cells) recombinantly expressing a HER2 TCR disclosed herein (e.g., HER2 TCRT cell) .

In some embodiments, the immune effector cell provided herein is a T cell. The T cell can be a cytotoxic T cell, a helper T cell, or a gamma delta T, a CD4+/CD8+ double positive T cell, a CD4+T cell, a CD8+ T cell, a CD4/CD8 double negative T cell, a CD3+ T cell, a naive T cell, an effector T cell, a cytotoxic T cell, a helper T cell, a memory T cell, a regulator T cell, a Th0 cell, a Th1 cell, a Th2 cell, a Th3 (Treg) cell, a Th9 cell, a Th17 cell, a Thαβ helper cell, a Tfh cell, a stem memory TSCM cell, a central memory TCM cell, an effector memory TEM cell, an effector memory TEMRA cell, or a gamma delta T cell. In some embodiments, the T cell is a cytotoxic T cell. In some embodiments, the T cell is genetically engineered. In some embodiments, the T cells provided herein are isolated. In some embodiments, the T cells provided herein are substantially pure.

In some embodiments, genetically engineered cells provided herein are derived from cells isolated from a subject. As used herein, a genetically engineered cell that is “derived from” a source cell means that the genetically engineered cell is obtained by taking the source cell and genetically manipulating the source cell. The source cell can be from a natural source. For example, the source cell can be a primary cell isolated from a subject. The subject can be an animal or a human. The source cell can also be a cell that has undergone passages or genetically manipulation in vitro.

In some embodiments, genetically engineered cells provided herein are derived from cells isolated from a human. Immune effector cells (e.g., T cells) can be obtained from many sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, T cell lines available in the art can be used. In some embodiments, genetically engineered cells provided herein are derived from cells isolated from peripheral blood. In some embodiments, genetically engineered cells provided herein are derived from cells isolated from bone marrow. In some embodiments, genetically engineered cells provided herein are derived from cells isolated from peripheral blood mononuclear cells (PBMC) .

In some embodiments, genetically engineered cells provided herein are derived from cells differentiated in vitro from a stem or progenitor cell. In some embodiments, the stem or progenitor cell is selected from the group consisting of a T cell progenitor cell, a hematopoietic stem and progenitor cell, a hematopoietic multipotent progenitor cell, an embryonic stem cell, and an induced pluripotent cell. In some embodiments, genetically engineered cells provided herein are derived from cells differentiated in vitro from a T cell progenitor cell. In some embodiments, genetically engineered cells provided herein are derived from cells differentiated in vitro from a hematopoietic stem and progenitor cell. In some embodiments, genetically engineered cells provided herein are derived from cells differentiated in vitro from a hematopoietic multipotent progenitor cell. In some embodiments, genetically engineered cells provided herein are derived from cells differentiated in vitro from an embryonic stem cell. In some embodiments, genetically engineered cells provided herein are derived from cells differentiated in vitro from an induced pluripotent cell.

In some embodiments, provided herein are a population of cells comprising a cell disclosed herein. The cells disclosed herein can comprise a polynucleotide that encodes a polypeptide disclosed herein or recombinantly express a polypeptide disclosed herein. The polypeptide can be an anti-HER2 antibody or antigen-binding fragment, a HER2 CAR, or a HER2 TCR. In some embodiments, cells disclosed herein can further express the fusion proteins disclosed herein or comprise a polynucleotide encoding a fusion protein disclosed herein. The fusion protein can be any fusion protein disclosed herein. In some embodiments, the population of cells provided herein express a CAR disclosed herein and a fusion protein disclosed herein. In some embodiments, the population of cells provided herein comprise a first polynucleotide encoding a fusion protein provided herein, and a second polynucleotide encoding a CAR. In some embodiments, the genetically engineered immune effector cells provided herein express a TCR disclosed herein and a fusion protein disclosed herein. In some embodiments, the population of cells provided herein comprise a first polynucleotide encoding a fusion protein provided herein, and a second polynucleotide encoding a TCR. In some embodiments, the population of cells provided herein comprise a polynucleotide that comprises a first fragment encoding a CAR and a second fragment encoding a fusion protein. In some embodiments, the population of cells provided herein comprise a polynucleotide that comprises a first fragment encoding a TCR and a second fragment encoding a fusion protein. In some embodiments, the polynucleotide has the first fragment and the second fragment from N-terminus to the C-terminus. In some embodiments, the polynucleotide has the second fragment and the first fragment, from N-terminus to the C-terminus.

The population of cells can be a homogenous population of cells. The population of cells can be a heterogeneous population of cells. In some embodiments, the population of cells can be a heterogeneous population of cells comprising any combination of the cells disclosed herein. In some embodiments, the population of cells are derived from peripheral blood mononuclear cells (PBMC) , peripheral blood leukocytes (PBL) , tumor infiltrating lymphocytes (TIL) , cytokine-induced killer cells (CIK) , lymphokine-activated killer cells (LAK) , or marrow infiltrate lymphocytes (MILs) . In some embodiments, the population of cells provided herein are derived from PBMC. In some embodiments, the population of cells provided herein are derived from PBL. In some embodiments, the population of cells provided herein are derived from TIL. In some embodiments, the population of cells provided herein are derived from CIK. In some embodiments, the population of cells provided herein are derived from LAK. In some embodiments, the population of cells provided herein are derived from MILs. The population of cells can be genetically engineered to recombinantly expressing a polypeptide (e.g., an antibody or a CAR) disclosed herein. In some embodiments, provided herein are population of cells comprising a polynucleotide encoding a polypeptide (e.g., an antibody or a CAR) disclosed herein, or a vector having a polynucleotide disclosed herein. In some embodiments, provided herein are population of cells comprising a polynucleotide that encodes an anti-HER2 antibody or antigen-binding fragment disclosed herein. In some embodiments, provided herein are population of cells recombinantly expressing an anti-HER2 antibody or antigen-binding fragment disclosed herein. In some embodiments, provided herein are population of cells comprising a polynucleotide that encodes a HER2 CAR disclosed herein. In some embodiments, provided herein are population of cells recombinantly expressing a HER2 CAR disclosed herein (e.g., HER2 CART cell) . In some embodiments, provided herein are population of cells comprising a polynucleotide that encodes a HER2 TCR disclosed herein. In some embodiments, provided herein are population of cells recombinantly expressing a HER2 TCR disclosed herein (e.g., HER2 TCRT cell) .
5.7 Safety Switch

In some embodiments, the cells provided herein comprise a safety switch, for example, for selectively eliminating the cell as needed during or after treatment.

Adverse events in cell therapy, particularly during the reinfusion of cells into patients, may be minimized by transducing immune cells with a suicide gene, also known as a safety switch. These safety switches can be implemented using various mechanisms. Various safety switch systems may be employed. For example, a TET-OFF/ON system may be used to regulate the expression of a CAR or other therapeutic gene based on the presence or absence of tetracycline. Alternatively, an NFAT-on switch may restrict gene expression to activated immune cells, providing conditional control of therapeutic activity.

The safety switch described herein refers broadly to any genetic element or system that enables conditional elimination of the cells in response to external stimuli.

In some embodiments, the safety switch is a suicide polypeptide, which, when expressed on the surface of the therapeutic cell, can trigger antibody-mediated clearance mechanisms such as antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) upon binding to an external antibody (e.g., rituximab targeting CD20 epitopes) . For example, the therapeutic cells (e.g., CAR-T cells) may be engineered to express a suicide polypeptide, such suicide polypeptide may comprise one or more CD20-derived epitopes, that can be targeted by a clinically approved antibody (e.g., rituximab) . When the suicide polypeptide containing such epitope is expressed on the surface of a therapeutic cell, binding of rituximab to the R epitopes may trigger immune-mediated lysis of the cell. The safety switch may comprise one or more epitopes, each epitope may independently bind an antibody molecule. This facilitates effective clearance of the therapeutic cells via ADCC and/or CDC. Administration of rituximab to the patient can thus induce in vivo elimination of the engineered cells. This safety mechanism may be employed when treatment-related toxicities reach unacceptable levels or when continued presence of therapeutic cells poses a risk to the patient.

In some embodiments, the suicide polypeptide comprises an epitope specific for (i.e., specifically recognized by) a monoclonal antibody. These epitopes are also referred to herein as mAb-specific epitopes. Exemplary mAb-specific epitopes are disclosed in International Patent Publication No.WO2016/120216, which is incorporated herein in its entirety.

Several epitope-monoclonal antibody couples can be used to generate CARs comprising monoclonal antibody specific epitopes; in particular, those already approved for medical use, such as CD20 epitope/rituximab as a non-limiting example. Table 7 provides exemplary mimotope sequences that can be inserted into the extracellular domains of any one of the CARs and corresponding mAb of the disclosure.

Table 7: Exemplary of epitopes and mimotopes

In some embodiments, the safety switch is included in the CAR construct.

In some embodiments, the extracellular domain of any one of the CARs disclosed herein may comprise one or more epitopes specific for (i.e., specifically recognized by) a monoclonal antibody. In these embodiments, the extracellular domain of the CARs comprises antigen binding domains that specifically bind to tumor antigen and one or more epitopes that bind to one or more monoclonal antibodies (mAbs) . CARs comprising the mAb-specific epitopes can be single-chain or multi-chain.

In some embodiments, the extracellular binding domain of the CAR comprises the following sequence: V1-L-V2-L-Epitope1-L-; V1-L-V2-L-Epitope1-L-Epitope2-L-; V1-L-V2-L-Epitope1-L-Epitope2-L-Epitope3-L-; L-Epitope1-L-V1-L-V2; L-Epitope1-L-Epitope2-L-V1-L-V2; Epitope1-L-Epitope2-L-Epitope3-L-V1-L-V2; L-Epitope1-L-V1-L-V2-L-Epitope2-L; L-Epitope1-L-V1-L -V2-L-Epitope2-L-Epitope3-L-; L-Epitope 1-L-V1-L-V2-L-Epitope2-L-Epitope3-L-Epitope4-L; L-Epitope1-L-Epitope2-L-V1-L-V2-L-Epitope3-L-; L-Epitope1-L-Epitope2-L-V1-L -V2-L-Epitope3-L-Epitope4-L-; V1-L-Epitope1-L-V2; V1-L-Epitope1-L-V2-L-Epitope2-L; V1-L-Epitope1-L-V2-L-Epitope2-L-Epitope3-L; V1-L-Epitope1-L-V2-L-Epitope2-L-Epitope3-L-Epitope4-L; L-Epitope1-L-V1-L-Epitope2-L-V2; or, L-Epitope1-L-V1-L-Epitope2-L-V2-L-Epitope3-L wherein, V1 is VL and V2 is VH or V1 is VH and V2 is VL; L is a linker comprising glycine and serine residues, and each occurrence of L in the extracellular binding domain can be identical or different to other occurrence of L in the same extracellular binding domain, and each occurrence of L is independently from the others; and, preferably comprising an amino sequence of SEQ ID NO: 21, and Epitope 1, Epitope 2 and Epitope 3 are mAb-specific epitopes and can be identical or different, optionally having the sequences of any one in Table 7.

In some embodiments, the safety switch is independently expressed on the surface of the therapeutic cell. In some embodiments, the safety switch is separately expressed and not physically located within the CAR. For example, the safety switch may be co-expressed with the CAR, either via a single promoter in an operably linked manner (e.g., using a self-cleaving peptide such as P2A or T2A) , or via a dual-promoter system enabling parallel expression of the CAR and the safety switch from the same vector.

In some embodiments, wherein the therapeutic cells (e.g., CAR-T cells) may further comprises a LACO described herein, the safety switch and the LACO can be co-expressed. In some embodiments, the cell described herein expresses the CAR, the LACO, and the safety switch as described in the present application. In some embodiments, the cell may further comprise one or more additional nucleic acid molecules selected from: a nucleic acid encoding the CAR described herein, a nucleic acid encoding the LACO described herein, and a nucleic acid encoding the safety switch described herein. The nucleic acids may be present on the same or separate vectors. The CAR, LACO, and safety switch may be expressed under the control of the same or different promoters, and may be linked via self-cleaving peptide sequences (e.g., T2A) or expressed as separate transcriptional units.

In some embodiments, the therapeutic cells are engineered using two separate vectors, wherein one vector encodes the CAR, and the other vector encodes the safety switch and an immunostimulatory molecule, such as LACO.

In addition, the safety switch may comprise other structural domains that facilitate its correct localization and expression on the cell membrane. These domains may include, but are not limited to, a signal peptide, a hinge region, and/or a transmembrane domain.

In some embodiments, the safety switch may comprise a signal peptide, one or more mAb-specific epitopes, a hinge region and/or a transmembrane domain. In some embodiments, the safety switch may comprise a signal peptide, one or more monoclonal antibody (mAb) -specific epitopes, a hinge region, and/or a transmembrane domain. The multiple mAb-specific epitopes, or the individual components of the safety switch, may be directly or indirectly linked, for example via linker sequences. In certain embodiments, the safety switch comprises, in sequence: a CD8α signal peptide, linker 1, a first R epitope, linker 2, a second R epitope, linker 3, a CD8 hinge region, and a CD8 transmembrane domain. The linker 1, linker 2, and linker 3 may be identical (e.g., SEQ ID NO: 21) or different. Similarly, the first and second R epitopes may be identical or distinct. In some embodiments, each R epitope is selected from known antibody-binding sequences, such as those listed in Table 7 (e.g., Rituximab binding mimotope as set forth in SEQ ID NO: 104) .

In some embodiments, the safety switch is co-expressed with LACO, and the amino acid sequence of the resulting fusion is set forth as SEQ ID NO: 105.

In another aspect, the present application also provides a nucleic acid molecule encoding the safety switch described herein, a vector comprising the nucleic acid molecule, and a cell that expresses the safety switch and/or contains the nucleic acid molecule and/or the vector.
5.8 Pharmaceutical Compositions

Provided herein are also pharmaceutical compositions comprising the anti-HER2 antibodies or antigen-binding fragments disclosed herein and/or the cells (e.g., CAR-T cells) disclosed herein. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the anti-HER2 antibodies or antigen-binding fragments disclosed herein and a pharmaceutically acceptable carrier. The anti-HER2 antibodies or antigen-binding fragments can be present at various concentrations. In some embodiments, the pharmaceutical compositions provided herein comprise soluble anti-HER2 antibodies or antigen-binding fragments provided herein at 1-1000 mg/ml. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the cells disclosed herein and a pharmaceutically acceptable carrier. The pharmaceutical compositions comprising the cells (e.g., CAR-T cells) disclosed herein can comprise a purified population of cells. Those skilled in the art can readily determine the percentage of cells in a cell population using various well-known methods, as described herein. The ranges of purity in cell populations comprising genetically engineered cells provided herein can be from about 20%to about 100%.

In some embodiments, the pharmaceutical compositions are useful in immunotherapy. In some embodiments, the pharmaceutical compositions are useful in immuno-oncology. In some embodiments, the pharmaceutical compositions are useful in inhibiting tumor growth in a subject (e.g., a human patient) . In some embodiments, the pharmaceutical compositions are useful in treating cancer in a subject (e.g., a human patient) .

Provided herein are also kits for preparation of pharmaceutical compositions having the anti-HER2 antibodies or antigen-binding fragments disclosed herein. In some embodiments, the kit comprises the anti-HER2 antibodies or antigen-binding fragments disclosed herein and a pharmaceutically acceptable carrier in one or more containers. In another embodiment, the kits can comprise anti-HER2 antibodies or antigen-binding fragments disclosed herein for administration to a subject. In specific embodiments, the kits comprise instructions regarding the preparation and/or administration of the anti-HER2 antibodies or antigen-binding fragments.

Provided herein are also kits for preparation of the cells (e.g., T cells) disclosed herein. In some embodiments, the kits comprise one or more vectors for generating a genetically engineered cell, such as a T cell, that expresses the anti-HER2 antibodies or antigen-binding fragments and/or the anti-HER2 CAR disclosed herein. The kits can be used to generate genetically engineered immune effector cells (e.g., T cells) from autologous or non-autologous cells to be administered to a compatible subject. In another embodiment, the kits can comprise immune effector cells disclosed herein for administration to a subject. In specific embodiments, the kits comprise the immune effector cells disclosed herein in one or more containers. In specific embodiments, the kits comprise instructions regarding the preparation and/or administration of the immune effector cells.

In some embodiments, provided herein is a pharmaceutical composition comprising anti-HER2 antibodies or antigen-binding fragments or cells provided herein wherein the composition is suitable for local administration. In some embodiments, local administration comprises intratumoral injection, peritumoral injection, juxta-tumoral injection, intralesional injection and/or injection into a tumor draining lymph node, or essentially any tumor-targeted injection where the antitumor agent is expected to leak into primary lymph nodes adjacent to targeted solid tumor.

Pharmaceutically acceptable carriers that can be used in compositions provided herein include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. In some embodiments, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion) . Depending on the route of administration, the active ingredient (i.e., anti-HER2 antibodies or antigen-binding fragments or immune effector cells provided herein) , can be coated in a material to protect the active ingredient from the action of acids and other natural conditions that can inactivate the active ingredient.

Provided herein are also pharmaceutical compositions or formulations that improve the stability of the anti-HER2 antibodies or antigen-binding fragments to allow for their long-term storage. In some embodiments, the pharmaceutical composition or formulation is stable for at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 1 year, at least 2 years, at least 3 years, at least 5 years or more. In some embodiments, the pharmaceutical composition or formulation is stable when stored at 4℃, 25℃, or 40℃.

The pharmaceutical compositions disclosed herein can further comprise one or more of a buffer system, a preservative, a tonicity agent, a chelating agent, a stabilizer, an antioxidant, a wetting agent, an emulsifying agent, a dispersing agent and/or a surfactant, as well as various combinations thereof. The use of preservatives, isotonic agents, chelating agents, stabilizers and surfactants in pharmaceutical compositions is well-known to the skilled person. Reference may be made to Remington: The Science and Practice of Pharmacy, 19th edition, 1995.

In some embodiments, the pharmaceutical composition is an aqueous formulation. Such a formulation is typically a solution or a suspension, but can also include colloids, dispersions, emulsions, and multi-phase materials. In some embodiments, the pharmaceutical compositions disclosed herein are freeze-dried, to which the physician or the patient adds solvents and/or diluents prior to use.

The amount of active ingredient which can be combined with a carrier material in the pharmaceutical compositions or formulations disclosed herein can vary. In some embodiments, the amount of active ingredient which can be combined with a carrier material is the amount that produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 0.01 percent to about ninety-nine percent of active ingredient, from about 0.1 percent to about 70 percent, or from about 1 percent to about 30 percent of active ingredient in combination with a pharmaceutically acceptable carrier.

The pharmaceutical compositions disclosed herein can be prepared with carriers that protect the active ingredient against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems.

In some embodiments, the anti-HER2 antibodies or antigen-binding fragments or immune effector cells (e.g., T cells) described herein can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that the activate ingredient described herein cross the BBB (if desired, e.g., for brain cancers) , they can be formulated, for example, in liposomes.
5.9 Methods and Uses

The present disclosure also provides methods of uses of the anti-HER2 antibodies or antigen-binding fragments, HER2 CARs, HER2 TCRs, polynucleotides encoding such anti-HER2 antibodies or antigen-binding fragments and HER2 CARs/TCRs, vectors comprising such polynucleotides, HER2 CAR/TCR-expressing cells or pharmaceutical compositions having such cells disclosed herein in treating cancer. Without being bound by theory, the anti-HER2 antibodies or antigen-binding fragments and the HER2 CAR/TCR-expressing cells disclosed herein can specifically target HER2 expressing cancer cells in vivo, thereby delivering their therapeutic effect of eliminating, lysing and/or killing cancer cells. In some embodiments, the methods include administering a therapeutically effective amount of the anti-HER2 antibodies or antigen-binding fragments disclosed herein to a subject in need thereof. In some embodiments, the methods include administering a therapeutically effective amount of HER2 CAR-expressing immune effector cells disclosed herein to a subject in need thereof. In one embodiment, the methods can include administering a therapeutically effective amount of HER2 CARTs disclosed herein to a subject in need thereof.

In some embodiments, provided herein are methods of treating tumor or cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the anti-HER2 antibodies or antigen-binding fragments disclosed herein. In some embodiments, provided herein are uses of the anti-HER2 antibodies or antigen-binding fragments disclosed herein in the treatment of tumor or cancer. In some embodiments, provided herein are uses of the anti-HER2 antibodies or antigen-binding fragments provided herein for the preparation of a medicament for the treatment of tumor or cancer.

In some embodiments, provided herein are methods of treating tumor or cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the immune effector cells (e.g., HER2 CARTs) disclosed herein. In some embodiments, provided herein are uses of the immune effector cells disclosed herein (e.g., HER2 CARTs) in treatment of tumor or cancer. In some embodiments, provided herein are uses of the immune effector cells (e.g., HER2 CARTs) provided herein for the preparation of a medicament for the treatment of tumor or cancer. In some embodiments, a population of cells comprising the immune effector cell disclosed herein is used in the treatment. The population of cells can be homogenous. The population of cells can be heterogenous.

In some embodiments, provided herein are methods of treating tumor or cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition disclosed herein. In some embodiments, provided herein are uses of the pharmaceutical composition disclosed herein in treatment of tumor or cancer. In some embodiments, provided herein are uses of the pharmaceutical composition provided herein for the preparation of a medicament for the treatment of tumor or cancer.

Actual dosage levels of the active ingredients (i.e., the anti-HER2 antibodies or antigen-binding fragments or the immune effector cells provided herein) in the pharmaceutical compositions described herein can be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions described herein, the route of administration, the time of administration, the rate of excretion, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

The anti-HER2 antibodies or antigen-binding fragments can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the anti-HER2 antibodies or antigen-binding fragments in the patient. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and until the patient shows partial or complete amelioration of symptoms of disease.

In some embodiments, immune effector cells provided herein that recombinantly express the HER2 CARs or TCRs disclosed herein can be used in the therapeutic methods disclosed herein. When a cell therapy is adopted, the cells provided herein can be administered as a dose based on cells per kilogram (cells/kg) of body weight of the subject to which the cells are administered. The cell doses can be in the range of about 10⁴ to about 10¹⁰ cells/kg of body weight, for example, about 10⁵ to about 10⁹, about 10⁵ to about 10⁸, about 10⁵ to about 10⁷, or about 10⁵ to 10⁶ cells/kg of body weight, depending on the mode and location of administration. In general, in the case of systemic administration, a higher dose is used than in regional administration, where the immune effector cells are administered in the region of a tumor. The precise determination of what would be considered an effective dose can be based on factors individual to each subject, including their size, age, sex, weight, and condition of the particular subject, as described above. Dosages can be readily determined by those skilled in the art based on the disclosure herein and knowledge in the art.

The anti-HER2 antibodies or antigen-binding fragments, immune effector cells, and pharmaceutical compositions provided herein can be administered to a subject by any methods known in the art, including, but not limited to, pleural administration, intravenous administration, subcutaneous administration, intranodal administration, intratumoral administration, intramuscular administration, intradermal administration, intrathecal administration, intrapleural administration, intraperitoneal administration, intracranial administration, spinal or other parenteral routes of administration, for example by injection or infusion, or direct administration to the thymus. The phrase “parenteral administration” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrastemal injection and infusion. In some embodiments, subcutaneous administration is adopted. In some embodiments, intravenous administration is adopted. In some embodiments, oral administration is adopted. In one embodiment, the cells provided herein can be delivered regionally to a tumor using well known methods, including but not limited to, hepatic or aortic pump; limb, lung or liver perfusion; in the portal vein; through a venous shunt; in a cavity or in a vein that is nearby a tumor, and the like. In another embodiment, the cells provided herein can be administered systemically. In a preferred embodiment, the cells are administered regionally at the site of a tumor. The cells can also be administered intratumorally, for example, by direct injection of the cells at the site of a tumor and/or into the tumor vasculature. For example, in the case of malignant pleural disease, mesothelioma or lung cancer, administration is preferably by intrapleural administration (see Adusumilli et al., Science Translational Medicine 6 (261) : 261ra151 (2014)) . One skilled in the art can select a suitable mode of administration based on the type of cancer and/or location of a tumor to be treated. The cells can be introduced by injection or catheter. In one embodiment, the cells are pleurally administered to the subject in need, for example, using an intrapleural catheter. Optionally, expansion and/or differentiation agents can be administered to the subject prior to, during or after administration of cells to increase production of the cells provided herein in vivo.

Proliferation of the cells provided herein is generally done ex vivo, prior to administration to a subject, and can be desirable in vivo after administration to a subject (see Kaiser et al., Cancer Gene Therapy 22: 72-78 (2015) ) . Cell proliferation should be accompanied by cell survival to permit cell expansion and persistence, such as with T cells.

Diseases that can be treated using the anti-HER2 antibodies or antigen-binding fragments, the immune effector cells, or the pharmaceutical compositions provided herein include any disease or disorder associated with HER2, and any disease or disorder in which HER2 is specifically expressed and/or in which HER2 has been targeted for treatment (collectively “HER2-associated diseases or disorders” ) . Ovarian, for example, is a disease associated with HER2.

In some embodiments, the anti-HER2 antibodies or antigen-binding fragments, cells, or pharmaceutical compositions provided herein can be used to treat ovarian cancer. Ovarian cancer is classified according to the histology of the tumor. Surface epithelial-stromal tumor, also known as ovarian epithelial carcinoma, is the most common type of ovarian cancer. It includes serous tumor (including serous papillary cystadenocarcinoma) , endometrioid tumor and mucinous cystadenocarcinoma.

The methods described herein can be used to treat various stages of ovarian cancer, e.g., stage I, stage II, stage III or stage IV. Staging can be performed, e.g., when the ovarian cancer is removed. Ovarian cancer is staged as follows: Stage I cancer is confined to one or both ovaries. The cancer is stage II if either one or both of the ovaries is involved and has spread to the uterus and/or the fallopian tubes or other sites in the pelvis. The cancer is stage III cancer if one or both of the ovaries is involved and has spread to lymph nodes or other sites outside of the pelvis but is still within the abdominal cavity, such as the surface of the intestine or liver. The cancer is stage IV cancer if one or both ovaries are involved and the cancer has spread outside the abdomen or to the inside of the liver.

In some embodiments, the ovarian cancer is resistant to one or more chemotherapeutic agent. In some embodiments, the ovarian cancer is refractory to the one or more chemotherapeutic agent.

Other cancers that can be treated with the anti-HER2 antibodies or antigen-binding fragments, cells, or pharmaceutical compositions provided herein include, e.g., breast cancer, gastric cancer, cervical cancer, urothelial cancer, esophageal cancer, bladder cancer, colorectal cancer, endometrial cancer, kidney cancer, lung cancer, pancreatic cancer, head and neck cancer, sarcoma, glioblastoma, prostate cancer, and/or thyroid cancer, and any combination thereof.

In certain diseases and conditions, HER2 is expressed on malignant cells and cancers. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a hematopoietic cancer. In some embodiments, the cancer is a renal cancer, lung cancer, urothelial cancer, breast cancer, brain tumor, thyroid cancer, leukemia, lymphoma, and/or multiple myeloma. In some embodiments, the cancer is renal cell carcinoma, non-small cell lung cancer, lung adenocarcinoma, glioblastoma, anaplastic thyroid cancer, B-cell non-Hodgkin’s lymphoma, and/or chronic lymphoblastic leukemia.

In some embodiments, the methods include identifying a subject who has, is suspected to have, or is at risk for developing a HER2-associated disease or disorder. Hence, provided are methods for identifying subjects with diseases or disorders associated with elevated HER2 expression and selecting them for treatment with the anti-HER2 antibodies or antigen-binding fragments, the immune effector cells, or the pharmaceutical compositions provided herein.

In some embodiments, a subject can be screened for the presence of a disease or disorder associated with elevated HER2 expression, such as a HER2-expressing cancer. In some embodiments, the methods include screening for or detecting the presence of a HER2-associated disease, e.g., a tumor. Thus, in some embodiments, a sample can be obtained from a patient suspected of having a disease or disorder associated with elevated HER2 expression and assayed for the expression level of HER2. In some embodiments, a subject who tests positive for a HER2-associated disease or disorder can be selected for treatment by the present methods, and can be administered a therapeutically effective amount of the anti-HER2 antibodies or antigen-binding fragments, the immune effector cells, or the pharmaceutical compositions provided herein.

In cancer treatment, eliminating cancer or tumor cells in a subject can occur, but any clinical improvement constitutes a benefit. An anti-tumor effect can be manifested by a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, or amelioration of various physiological symptoms associated with the cancerous condition. An anti-tumor effect can also be manifested by the ability of the cells or pharmaceutical compositions provided herein in prevention of the occurrence of tumor in the first place. In some embodiments, an “anti-tumor effect” can be manifested by the reduction in cancer-induced immunosuppression. Clinical improvement comprises decreased risk or rate of progression or reduction in pathological consequences of the cancer or tumor. It is also understood that a method of treating cancer can include any effect that ameliorates a sign or symptom associated with cancer. Such signs or symptoms include, but are not limited to, reducing tumor burden, including inhibiting growth of a tumor, slowing the growth rate of a tumor, reducing the size of a tumor, reducing the number of tumors, eliminating a tumor, all of which can be measured using routine tumor imaging techniques well known in the art. Other signs or symptoms associated with cancer include, but are not limited to, fatigue, pain, weight loss, and other signs or symptoms associated with various cancers.

In some embodiments, the methods or uses provided herein can reduce tumor burden. Thus, administration of the anti-HER2 antibodies or antigen-binding fragments, cells or pharmaceutical compositions disclosed herein can reduce the number of tumor cells, reduce tumor size, and/or eradicate the tumor in the subject. Methods for monitoring patient response to administration of a pharmaceutical composition disclosed herein are known in the art and can be employed in accordance with methods disclosed herein.

In the methods disclosed herein, a therapeutically effective amount of the anti-HER2 antibodies or antigen-binding fragments, cells or pharmaceutical compositions disclosed herein is administered to a subject in need of cancer treatment. The subject can be a mammal. In some embodiments, the subject is a human. In some embodiments, these individuals have no clinically measurable tumor. However, they are suspected of being at risk for progression of the disease, either near the original tumor site, or by metastases. This group can be further subdivided into high-risk and low-risk individuals. The subdivision is made on the basis of features observed before or after the initial treatment. These features are known in the clinical arts and are suitably defined for different types of cancers. Features typical of high-risk subgroups are those in which the tumor has invaded neighboring tissues, or who show involvement of lymph nodes.

In some embodiments, the subject has persistent or relapsed disease, e.g., following treatment with another HER2-specific antibody and/or HER2 CART and/or other therapy, including chemotherapy, radiation, and/or hematopoietic stem cell transplantation (HSCT) , e.g., allogeneic HSCT or autologous HSCT. In some embodiments, the administration effectively treats the subject despite the subject having become resistant to another HER2-targeted therapy. In some embodiments, the subject has not relapsed but is determined to be at risk for relapse, such as at a high risk of relapse, and thus the compound or composition is administered prophylactically, e.g., to reduce the likelihood of or prevent relapse.

In some embodiments, the subject is one that is eligible for a transplant, such as is eligible for a hematopoietic stem cell transplantation (HSCT) , e.g., allogeneic HSCT or autologous HSCT. In some embodiments, the subject has not previously received a transplant, despite being eligible, prior to administration of the HER2-binding molecules, including the anti-HER2 antibodies or antigen-binding fragments, the immune effector cells, or the pharmaceutical compositions provided herein. In some embodiments, the subject is one that is not eligible for a transplant, such as is not eligible for a hematopoietic stem cell transplantation (HSCT) , e.g., allogenic HSCT or autologous HSCT.

In some embodiments, the methods provided herein include adoptive cell therapy, whereby genetically engineered immune effector cells expressing the provided recombinant receptors comprising a HER2-binding molecule (e.g., HER2 CARs provided herein) are administered to subjects. Such administration can promote activation of the cells (e.g., T cell activation) in a HER2-targeted manner, such that the cells of the disease or disorder are targeted for destruction. Thus, the provided methods and uses include methods and uses for adoptive cell therapy. In some embodiments, the methods include administration of the cells or a composition containing the cells to a subject such as one having, or at risk for, or suspected of having the disease, condition or disorder. In some embodiments, the cells, populations, and compositions are administered to a subject having the particular disease or condition to be treated, e.g., via adoptive cell therapy, such as adoptive T cell therapy. In some embodiments, the cells or compositions are administered to the subject, such as a subject having or at risk for the disease or condition. In some aspects, the methods thereby treat, e.g., ameliorate one or more symptom of the disease or condition, such as by lessening tumor burden in a HER2-expressing cancer.

Methods for administration of cells for adoptive cell therapy are known and can be used in connection with the provided methods and compositions. For example, adoptive T cell therapy methods are described, e.g., in US Patent Application Publication No. 2003/0170238 to Gruenberg et al.; US Patent No. 4, 690, 915 to Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8 (10) : 577-85) . See, e.g., Themeli et al. (2013) Nat Biotechnol. 31 (10) : 928-933; Tsukahara et al. (2013) Biochem Biophys Res Commun 438 (1) : 84-9; Davila et al. (2013) PLoS ONE 8 (4) : e61338.

In some embodiments, the cell therapy, e.g., adoptive cell therapy, e.g., adoptive T cell therapy, is carried out by autologous transfer, in which the cells are isolated and/or otherwise prepared from the subject who is to receive the cell therapy, or from a sample derived from such a subject. Thus, in some embodiments, the cells are derived from a subject, e.g., patient, in need of a treatment and the cells, following isolation and processing are administered to the same subject.

In some embodiments, the cell therapy, e.g., adoptive cell therapy, e.g., adoptive T cell therapy, is carried out by allogeneic transfer, in which the cells are isolated and/or otherwise prepared from a subject other than a subject who is to receive or who ultimately receives the cell therapy, e.g., a first subject. In such embodiments, the cells then are administered to a different subject, e.g., a second subject, of the same species. In some embodiments, the first and second subjects are genetically identical. In some embodiments, the first and second subjects are genetically similar. In some embodiments, the second subject expresses the same HLA class or supertype as the first subject.

Anti-HER2 antibodies or antigen-binding fragments, cells, or pharmaceutical compositions provided herein can be administered with medical devices known in the art. For example, in some embodiments, a needleless hypodermic injection device can be used, such as the devices disclosed in U.S. Patent Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or 4,596,556. Examples of well-known implants and modules for use described herein include: U.S. Patent No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Patent No. 4,486,194, which discloses a therapeutic device for administering medicaments through the skin; U.S. Patent No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Patent No. 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Patent No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments; and U.S. Patent No. 4,475,196, which discloses an osmotic drug delivery system. These patents are incorporated herein by reference. Many other such implants, delivery systems, and modules are known to those skilled in the art.

Combination therapy using agents with different mechanisms of action can result in additive or synergetic effects. Combination therapy can allow for a lower dose of each agent than is used in monotherapy, thereby reducing toxic side effects and/or increasing the therapeutic index of the agent disclosed herein. Combination therapy can decrease the likelihood that resistant cancer cells will develop. In some embodiments, the additional therapy results in an increase in the therapeutic index of the cells or pharmaceutical compositions described herein. In some embodiments, the additional therapy results in a decrease in the toxicity and/or side effects of cells or pharmaceutical compositions described herein. In some embodiments, the anti-HER2 antibodies or antigen-binding fragments, cells, or pharmaceutical compositions described herein can be administered in combination with an additional therapy. In some embodiments, the additional therapy can be surgical resection, radiotherapy, or chemotherapy.

The additional therapy can be administered prior to, concurrently with, or subsequent to administration of the anti-HER2 antibodies or antigen-binding fragments, cells, or pharmaceutical compositions described herein. Combined administration can include co-administration, either in a single pharmaceutical formulation or using separate formulations, or consecutive administration in either order but generally within a time period such that all active agents can exert their biological activities simultaneously. A person skilled in the art can readily determine appropriate regimens for administering a pharmaceutical composition described herein and an additional therapy in combination, including the timing and dosing of an additional agent to be used in a combination therapy, based on the needs of the subject being treated.
5.10 Methods of production

The following examples are set forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc. ) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., bp, base pair (s) ; kb, kilobase (s) ; pl, picoliter (s) ; s or sec, second (s) ; min, minute (s) ; h or hr, hour (s) ; aa, amino acid (s) ; nt, nucleotide (s) ; i.m., intramuscular (ly) ; i.p., intraperitoneal (ly) ; s.c., subcutaneous (ly) ; and the like.

Materials and Methods

Primary human lymphocytes. Primary human CD4+ T cells and CD8+ T cells were isolated from healthy volunteer donors. T cells were stimulated with anti-CD3/CD28 Dynabeads (Life Technologies, Grand Island, NY) and cultured in the R10 medium (RPMI-1640 medium supplemented with 10%fetal bovine serum, 1%HEPES, 1%GutaMAX, 1%penicillin and streptomycin, 1%MEM NEAA, and 1%sodium pyruvate) supplemented with 100 IU/mL IL-2.

Cell lines. The following cell lines were cultured in R10 medium and used in related experiments: SKOV3 (human ovarian cancer cells) , A549 (human lung cancer cells) , SY5Y (human neuroblastoma cells) .

Lentivirus production and transduction. Lentiviral particles were produced from HEK293T cells transfected with transfer plasmids and packaging plasmids pRSV. REV, pMD2. G and pMDLg. pRRE. Lentiviral particles were harvested after 24 and 48 hours and concentrated by ultracentrifugation.

T cells were stimulated by CD3/CD28 dynabeads on day 0. Lentivirus was added to culture medium on day 1. The cells were fed with R10 medium with 100 IU/ml IL-2 every day or every two days.

Production of anti-HER2 CAR mRNA. In vitro transcription of mRNA was performed using the Ambion mMessage mMACHINE T7 Ultra kit (Life Technologies, Carlsbad, CA) .

Electroporation of anti-HER2 CAR mRNA. mRNA was added to 0.1 ml T cells (0.5×108 cells/ml) that were washed twice and resuspended with OPTI-MEM. Electroporation was performed in a 0.2 cm cuvette with a BTX830 (Harvard Apparatus BTX) at 500 V and 0.7 ms.

Tumor killing assay using IncuCyte real-time cell analyzer. Tumor cells and T cells were washed with R10 medium twice, and then were resuspended in R10 medium. Tumor cells were seeded at 3, 000 cells/well and CAR+ T cells were added according to effect-target ratiosin a 96 well flat-bottom plate. Total integrated intensity was recorded every 8 hours or longer time.

ELISA. 0.1 million of CAR-T cells and 0.1 million of tumor cells were co-cultured at 37 ℃for 24 h, then the supernatant was collected and the ELISA assay was performed to measure IL-2 and IFN-γ secretion levels following the instruction manual of ELISA kits (R&D Systems) .
5.11 Examples
5.11.1 Example 1: Phage display library screening of Anti-HER2 antibody

According to the initial OD value of about 0.1, inoculate an appropriate amount of original phage library liquid into a 500ml 2×YT medium Erlenmeyer flask, and add 100 μg/mL ampicillin and 2%glucose. 250rpm, 37℃, shake the bacteria for about 3 hours until the OD600 value is about 0.5.

Add about 3×10¹² M13KO7 helper phage to the above bacterial solution and let it stand at 37 degrees for 30 minutes.

After 30 minutes, transfer the infected bacterial solution to a 50ml centrifuge tube, centrifuge at 2400g, 12℃ for 30 minutes, and discard the supernatant to remove the inhibitory effect of glucose on antibody expression.

Resuspend the pellet to 600 ml 2×YT, and add ampicillin, 60 μg/mL kanamycin, and 100 μM IPTG at a final concentration of 100 μg/mL) . Shake culture overnight at 30℃, 200 rpm.

The next day, collect the bacterial liquid, centrifuge at 2400g, 12℃ for 30 minutes.

After centrifugation, transfer 36ml of the supernatant to a new 50ml centrifuge tube, then add 9ml of PEG/NaCl, mix thoroughly, and place on ice for 1-2 hours. After 1-2 hours, centrifuge the mixture at 4000 rpm and 4℃ for 30 minutes.

After centrifugation, gently discard the supernatant, resuspend the phage pellet in 3 ml of 1×PBS/5%FBS solution, and store it at 4 degrees for later use.

One day before the first round of solid-phase panning, use a flat-bottomed 96-well plate to coat HER2-6His protein at a coating concentration of 10 μg/ml, and keep the coating overnight in a refrigerator at 4 degrees Celsius.

Incubate the purified phage library and coated HER2 antigen for 2 hours. After 2 hours, wash with 0.05%Tween-20/PBS and remove as much supernatant as possible.

Use 500 μl of 0.1M, pH2.2 Glycine to elute the phage and let it stand at room temperature for 10 minutes. After centrifugation, transfer the supernatant to a new tube, add 50ul pH9.5 Tris-HCL to neutralize the pH to neutral, and then transfer the supernatant to a new 15ml tube for infecting TG1.

Repeat the panning operation twice more, and the clones finally obtained are tested by phage ELISA (the results are shown in FIG. 1) , and the positive clones are amplified by PCR to obtain the scFv sequence (the gray background in FIG. 1 is the PCR identified clone) .
5.11.2 Example 2: Construction of anti-HER2 CAR vector and in Vitro Cytotoxicity Assay on
Anti-HER2 CAR-T Cells

2.1 Construction of anti-HER2 CAR vector for mRNA production

The pGEM vector was digested with EcoRI and SalI enzymes and purified by gel purification. The anti-HER2 single chain antibody (scFv) sequence and CAR fragment (from hinge domain to CD3ζ domain) were amplified by PCR, digested with XhoI and EcoRI, and purified by gel purification. The single chain antibody fragment, CAR fragment (from hinge domain to CD3ζ domain) and pGEM vector were ligated with T4 ligase and transformed into competent cells. Correct colonies were selected for further experiments after being confirmed by sanger sequencing.

2.2 In vitro transcription (IVT) of CAR mRNA

The pGEM-CAR plasmid was digested with SpeI enzyme to be linearized. The linearized vector was purified using a PCR purification kit and eluted with RNase-free water. The concentration of DNA was determined using a nanodrop and verified by performing agarose gel electrophoresis for DNA. IVT was performed according to the protocol of the manufacturer (ThermoFisher, Cat No: AM13455) . Specifically, 1 μg of template DNA, NTP/ARCA buffer, T7 buffer, GTP, T7 enzyme mix and RNase-free H2O were added to a 0.2 mL PCR tube in a volume of 20 μL and incubated at 37 ℃ for 3 h. 3 h later, 2 μL of DNase was added to each reaction and incubated for 15 min at 37 ℃. The tailing procedure was then performed according to the manufacturer's recommendations. IVT mRNA was purified using an RNasy kit (Qiagen) . The concentration of RNA was determined using a nanodrop and verified by performing PAGE electrophoresis for DNA.

2.3 Preparation and characterization of anti-HER2 CAR-T cells

SKOV3 tumor cells and T cells were collected and washed 3 times with an Opti-MEM medium. The cell pellet was resuspended in an Opti-MEM medium at a cell concentration of 1×10e7/mL. 10 μg of anti-HER2 CAR mRNA was added to 100 μL of T cells and mixed well. The parameters on a BTX machine were set: for T cells: 500 V, 0.7 ms. 100 μL of cells mixed with RNA were added to the BTX electroporation cuvette, which was then tapped gently to avoid air bubbles. Electroporation was performed, and then the cells were transferred to a pre-heated medium and cultured at 37 ℃.

The binding of anti-HER2 CART cells to HER2-Fc recombinant protein, 082 and 072 was determined by FACS staining. As shown in FIG. 2, CAR-T cells (H13) expressing anti-HER2 scFv were able to bind to the HER2-Fc protein (FIG. 2A) and 082 (FIG. 2C) , but not to 072 (FIG. 2B) . This was because H13 scFv was of human rather than murine origin. wherein NO EP was a control T cell without a CAR molecule, m4D5 (SEQ ID NO: 107) , 4D5-5 (SEQ ID NO: 108) and m4D5-3 (SEQ ID NO: 109) were control CAR-T cells (Liu etc. Affinity-Tuned ErbB2 or EGFR Chimeric Antigen Receptor T Cells Exhibit an Increased Therapeutic Index against Tumors in Mice. Cancer Res 1 September 2015, 75 (17) : 3596-3607) .

2.4 In Vitro Cytotoxicity Assay on Anti-HER2 CAR-T Cells

SKOV3-CBG-GFP cells, which had a high expression of HER2 antigen, were seeded into a flat bottom 96-well plate at 3000 cells/100 μL/well. CAR-T cells were diluted to appropriate concentrations and seeded at 100 μL/well into tumor cells at different E/T ratios, e.g., 3: 1 or 1: 1. Meanwhile, A549-CBG-GFP cells, which slightly expressed HER2 protein, were as the control cells in this experiment. The co-culture plate was placed into the IncuCyte S3 machine and the scan parameters were set. After scanning for 3 days, the total green object integrated intensity (GCU ×μm²/Well) was analyzed to calculate the killing efficiency.

2.5 Cytokines assay

mRNA-based anti-HER2 CART cells incubated with SKOV3-CBG-GFP or A549-ESO-CBG-GFP tumor cells at E/T ratio=1: 1.24 hours later, the supernatant was used to detect cytokines IL-2 and IFN-γ. For SKOV3 cells, H13 had high levels of IL-2 (FIG. 5A) and IFN-γ (FIG. 5B) . This indicated that H13 could proliferate better and kill tumor cells with high expression of antigen. When m4D5 was co-incubated with A549, the secretion of IL-2 and IFN-γ was more obvious than other CAR-T cells (FIG. 5A and FIG. 5B) . This indicated that m4D5 had an overreaction to tumor cells that weakly expressed the antigen.

FIGs. 3A-3B showed the killing curves of anti-HER2 CAR-T cells against SKOV3-CBG-GFP tumor cells at an E/T ratio of 3: 1 (FIG. 3A) and 1: 1 (FIG. 3B) . FIGs. 3A-3B showed that anti-HER2 CAR-T cells (H13) could kill SKOV3 cells well.

FIGs. 4A-4B showed the killing curves of anti-HER2 CAR-T cells against A549-CBG-GFP tumor cells at an E/T ratio of 3: 1 and 1: 1. FIGs. 4A-4B showed that the killing effect of anti-HER2 CAR-T cells (H13) on A549 tumor cells slightly expressing HER2 is significantly weaker than m4D5.
5.11.3 Example 3: Preparation of lentiviral CAR-T cells

The H13 HER2 CAR sequence and HER2 CAR/LACOSTIM sequences were constructed into the pUTCK vector respectively (as shown in FIG. 6 and FIG. 12) . Then, perform plasmid extraction, lentivirus packaging, ultracentrifugation, and titer determination. Finally, freeze them in a -80℃ refrigerator for later use. Preparation of CAR-T cells: On day 0, activate T cells with anti-CD3/CD28 Dynabeads, and adjust the cell density to 1e6 cells/ml. On day 1, add lentivirus to T cells at an MOI of 3 and culture for another two days. On day 5, remove the anti-CD3/CD28 Dynabeads, perform cell counting, passage and rehydration. On day 9, take 2e5 T cells from each group, stain them with HER2-Fc/anti-human IgG Fc and CD40-Fc/anti-human IgG Fc antibodies respectively, conduct flow cytometry analysis to detect the CAR positivity rate (as shown in FIG. 7 and FIG. 13) , and assess the expression of LACOSTIM (as shown in FIG. 8 and FIG. 14) .

Continue to culture the CAR-T cells until about day 13, harvest them, and use them directly for functional experiments, or freeze them for later use.
5.11.4 Example 4: In vitro cytotoxicity testing of CAR-T cell preparation

1. Twelve hours prior to co-culture for cytotoxicity experiments with CAR-T cells, seed tumor cells into a flat-bottom 96-well plate at a density of 3, 000 cells/100 μl per well.

2. After 8-12 hours, allow the cells to fully adhere to the well surface. Dilute various anti-HER2 CAR-T cells to appropriate cell densities and co-incubate them with tumor cells at different effector-to-target ratios (such as E: T=3: 1, 1: 1, and 0.3: 1) .

3. Place the 96-well plate into the InCucyte S3 machine and set the scanning parameters.

4. After 3 days of scanning, analyze the total green fluorescence cumulative intensity (GCU xμm²/well) to calculate tumor cell killing efficiency. The results are shown in FIG. 9-11 and FIG. 15-19. Compared with m4D5 cells, H13 CAR-T cells, and 3 types of HER2 CAR/LACOSTIM CAR-T cells, such as H13. A40517 (H13-A40517-CD28) , H13.25-9.3h11 (H13-A4025-9.3h11 ) and H13+R2.25-9.3h11 (H13 CAR and R2-A4025-9.3h11 LACO are expressed by different vector, the amino acid sequence of R2-A4025-9.3h11 LACO is as set forth in SEQ ID NO: 105, the R in R2-A4025-9.3h11 is Rituximab binding mimotope) CAR-T cells, have minimal killing effect on A549 cells with low HER2 expression levels (see FIG. 10 and FIG. 16) .

Furthermore, for SY5Y that do not express HER2, was screened as target cells (see FIG. 11 and FIG. 19) . Various candidate sequences demonstrated good specificity.
5.11.5 Example 5: Detection of tumor suppression in tumor-bearing animal models in vivo

Conducting animal model experiments using HER2 CAR/LACOSTIM CAR-T cells (e.g. H13+25-9.3h11 CAR-T cells) . SKOV3 cells in the expansion phase were subcutaneously injected into immunodeficient M-NSG mice at a dose of 7×10⁶ cells per mouse to establish an SKOV3 subcutaneous tumor-bearing model. When tumor volumes reached 70-100 mm³, the tumor-bearing mice were divided into three groups according to Table 8 and administered CAR-T cells at the dose of 5×10⁵CAR-positive cells, respectively. The grouping regimens are detailed in Table 8.

Table 8 Grouping Scheme for Animal Model Experiments

The mice were continuously maintained under standardized conditions, with tumor volume, body weight, and the proportion of CD45+CD3+ cells recorded throughout the study. Results are shown in FIG. 21. The experimental data from the mouse model demonstrated that following a single injection of CAR-T cells, both the H13 and H13+LACO CAR-T cell groups exhibited significant tumor-killing efficacy compared to the NTD group. Compared with H13 group, the H13+LACO group could better control the tumor growth (showed in FIG. 21A) .

Detection of CAR-T Cells Change in Tumor-Bearing Animal Models. The proportion of CAR+ T cells in the peripheral blood of tumor-bearing mice was detected by flow cytometry after administration, and the expansion of CAR-T cells in the mice was analyzed. The results are shown in FIG. 21C. The results indicate that after a single injection of CAR-T cells, the proportion of CAR-T cells in the peripheral blood of H13+LACO group gradually increased, reaching a peak at day 10-15. This demonstrates that CAR-T cells undergo significant expansion during the process of tumor killing. Meanwhile H13+R2.25-9.3h11 group had a higher proportion of CART cells than H13. This suggests that LACO may promote the proliferation of CAR-T cells in the peripheral blood of tumor-bearing mice.

While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

An antibody or antigen-binding fragment thereof that specifically binds HER2, comprising a heavy chain variable region (VH) comprising a heavy chain CDR1 (HCDR1) , a heavy chain CDR2 (HCDR2) , a heavy chain CDR3 (HCDR3) having an amino acid sequence of SEQ ID NOs: 4, 5, and 6 respectively; or a variant thereof having up to about 5 amino acid substitutions, additions, and/or deletions in the HCDRs.
The antibody or antigen-binding fragment thereof of claim 1, further comprising:

a light chain variable region (VL) comprising a light chain CDR1 (LCDR1) , light chain CDR2 (LCDR2) , a light chain CDR3 (LCDR3) having an amino acid sequence of SEQ ID NOs: 1, 2, and 3 respectively; or a variant thereof having up to about 5 amino acid substitutions, additions, and/or deletions in the LCDRs.
The antibody or antigen-binding fragment thereof of claim 2, comprising a LCDR1, a LCDR2, a LCDR3, a HCDR1, a HCDR2 and a HCDR3, wherein

(a) the LCDR1, CDR2 and CDR3 have the amino acid sequences of SEQ ID NOs: 1, 2, and 3 respectively; and

(b) the HCDR1, CDR2 and CDR3 have the amino acid sequences of SEQ ID NOs: 4, 5, and 6 respectively.
The antibody or antigen-binding fragment thereof of any one of claims 1-3, comprising:

(a) a VL having at least 85%, at least 90%, at least 95%, at least 98%, or 100%sequence identity to an amino acid sequence of SEQ ID NO: 7; and/or

(b) a VH having at least 85%, at least 90%, at least 95%, at least 98%, or 100%sequence identity to an amino acid sequence of SEQ ID NO: 8.
The antibody or antigen-binding fragment thereof of any one of claims 1-4, comprising a VL and a VH, wherein the VL and the VH have the amino acid sequences of SEQ ID NOs: 7 and 8, respectively.
The antibody or antigen-binding fragment thereof of any one of claims 1-5, wherein the antibody or antigen-binding fragment is a monoclonal antibody or antigen-binding fragment.
The antibody or antigen-binding fragment thereof of any one of claims 1-6, wherein the antibody or antigen-binding fragment is selected from the group consisting of an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, and an IgG4 antibody.
The antibody or antigen-binding fragment thereof of any one of claims 1-7 that is selected from the group consisting of a Fab, a Fab’, a F (ab’) ₂, a Fv, a scFv, a (scFv) ₂, a single domain antibody (sdAb) , and a heavy chain antibody (HCAb) .
The antibody or antigen-binding fragment thereof of any one of claims 1-8 that is a scFv having the amino acid sequence of SEQ ID NO: 9.
A Chimeric Antigen Receptor (CAR) that specifically binds HER2, comprising, from N-terminus to C-terminus:

(a) a HER2-binding domain that comprises the antibody or antigen-binding fragment thereof of any one of claims 1-8;

(b) a transmembrane domain; and

(c) a cytoplasmic domain.
The CAR of claim 10, wherein the transmembrane domain is derived from CD8, CD28, CD3ζ, CD4, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, BTLA, TCR α chain, TCR β chain, or TCR ζ chain, CD3ε, CD45, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, or CD154.
The CAR of any one of claims 10-11, wherein the transmembrane domain comprises CD8 transmembrane region.
The CAR of any one of claims 10-12, wherein the cytoplasmic domain comprises a signaling domain derived from CD3ζ, FcRγ, FcγRIIa, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, DAP10, DAP12, or any combination thereof.
The CAR of any one of claims 10-13, wherein the cytoplasmic domain further comprises a co-stimulatory domain derived from CD28, 4-1BB (CD137) , OX40, ICOS, DAP10, 2B4, CD27, CD30, CD40, CD2, CD7, LIGHT, GITR, TLR, DR3, CD43, or any combination thereof.
The CAR of any one of claims 10-14, wherein the cytoplasmic domain comprises a CD3ζ signaling domain and a 4-1BB co-stimulatory domain.
The CAR of any one of claims 10-15, further comprising a CD8 hinge domain between the HER2-binding domain and the transmembrane domain.
The CAR of any one of claims 10-16, further comprising a signal peptide domain derived from CD8 at N-terminus.
The CAR of any one of claims 10-17, comprising an amino acid sequence of SEQ ID NO: 10.
A polynucleotide that encodes the antibody or antigen-binding fragment of anyone of claims 1-9 or the CAR of any one of claims 10-18.
The polynucleotide of claim 19, wherein the polynucleotide is a DNA or mRNA.
A vector that comprises the polynucleotide of claim 20.
A cell comprising the antibody or antigen-binding fragment of any one of claims 1-9, the CAR of any one of claims 10-18, the polynucleotide encoding the CAR of any one of claims 19-20, or the vector of claim 21.
The cell of claim 22, wherein the cell is an immune effector cell.
The cell of any one of claims 22-23, wherein the cell is derived from a cell isolated from peripheral blood or bone marrow.
The cell of any one of claims 22-24, wherein the cell is derived from a cell differentiated in vitro from a stem or progenitor cell is selected from the group consisting of a T cell progenitor cell, a hematopoietic stem and progenitor cell, a hematopoietic multipotent progenitor cell, an embryonic stem cell, and an induced pluripotent cell.
The cell of any one of claims 22-25, wherein the cell is a T cell or a NK cell.
The cell of any one of claims 22-26, wherein the cell is a cytotoxic T cell, a helper T cell, a gamma delta T, a CD4⁺/CD8⁺ double positive T cell, a CD4⁺ T cell, a CD8⁺ T cell, a CD4^-/CD8^- double negative T cell, a CD3⁺ T cell, a naive T cell, an effector T cell, a helper T cell, a memory T cell, a regulator T cell, a Th0 cell, a Th1 cell, a Th2 cell, a Th3 (Treg) cell, a Th9 cell, a Th17 cell, a Thαβ helper cell, a Tfh cell, a stem memory TSCM cell, a central memory TCM cell, an effector memory TEM cell, or an effector memory TEMRA cell.
The cell of any one of claims 22-27, wherein the cell recombinantly expresses a fusion protein comprising a first domain that activates an antigen-presenting cell (APC) and a second domain that activates an immune effector cell, wherein

(i) the first domain comprises (a) a ligand that binds an activation receptor of the APC, or a receptor-binding fragment thereof, or (b) an antibody that binds an activation receptor of the APC, or an antigen-binding fragment thereof; and

(ii) the second domain comprises (a) a co-stimulatory receptor of the immune effector cell, or a functional fragment thereof, (b) a co-stimulatory ligand of the immune effector cell, or a receptor-binding fragment thereof, or (c) an antibody that binds a co-stimulatory receptor of the immune effector cell, or an antigen-binding fragment thereof.
The cell of claim 28, wherein the N-terminus of the first domain is linked to the C-terminus of the second domain.
The cell of claim 28, wherein the N-terminus of the second domain is linked to the C-terminus of the first domain.
The cell of any one of claims 28-30, wherein the first domain and the second domain are linked via a linker.
The cell of any one of claims 28-31, wherein the APC is selected from the group consisting of a dendritic cell, a macrophage, a myeloid derived suppressor cell, a monocyte, a B cell, a T cell, and a Langerhans cell.
The cell of any one of claims 28-32, wherein the activation receptor of the APC is selected from the group consisting of CD40, CD80, CD86, CD91, DEC-205 and DC-SIGN.
The cell of claim 33, wherein the first domain comprises a ligand that binds CD40, CD80, CD86, CD91, DEC-205 or DC-SIGN, or a receptor-binding fragment thereof.
The cell of claim 34, wherein the first domain comprises a receptor-binding fragment of CD40 Ligand (CD40L) .
The cell of any one of claims 28-32, wherein the first domain comprises an antibody that binds the activation receptor of the APC, or an antigen-binding fragment thereof.
The cell of claim 36, wherein the first domain is an anti-CD40 antibody or an antigen-binding fragment thereof.
The cell of any one of claims 28-37, wherein the immune effector cell is selected from the group consisting of a T cell, an NK cell, an NKT cell, a macrophage, a neutrophil, and a granulocyte.
The cell of any one of claims 28-38 wherein the second domain comprises a cytoplasmic domain of the co-stimulatory receptor.
The cell of claim 39, wherein the co-stimulatory receptor is selected from the group consisting of CD28, 4-1BB, ICOS, CD27, OX40, DAP10, 2B4, CD30, CD2, LIGHT, GITR, TLR, DR3, and CD43.
The cell of claim 39, wherein the co-stimulatory receptor is CD28.
The cell of any one of claims 28-38, wherein the second domain is a co-stimulatory ligand of the immune effector cell, or a receptor-binding fragment thereof.
The cell of claim 42, wherein the co-stimulatory ligand is selected from the group consisting of CD58, CD70, CD83, CD80, CD86, CD137L, CD252, CD275, CD54, CD49a, CD112, CD150, CD155, CD265, CD270, TL1A, CD127, IL-4R, GITR-L, TIM-4, CD153, CD48, CD160, CD200R, and CD44.
The cell of any one of claims 28-38, wherein the second domain is an antibody that binds the co-stimulatory receptor, or an antigen-binding fragment thereof.
The cell of claim 44, wherein the second domain is an scFv.
The cell of claim 44 or 45, wherein the co-stimulatory receptor is selected from the group consisting of CD28, 4-1BB, ICOS, CD27, OX40, DAP10, 2B4, CD30, CD2, LIGHT, GITR, TLR, DR3, and CD43.
The cell of claim 44 or 45, wherein the co-stimulatory receptor is CD28.
The cell of claim 28, wherein the first domain comprises an anti-CD40 antibody or an antigen-binding fragment thereof, and the second domain comprises an anti-CD28 antibody or an antigen-binding fragment thereof.
The cell of claim 28, wherein the first domain comprises an anti-CD40 antibody or an antigen-binding fragment thereof, and the second domain comprises a CD28 cytoplasmic domain.
The cell of any one of claim 28-49, wherein the fusion protein is at least 85%, 90%, 95%, 98%, 99%, or 100%identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 50-61 and 65-76.
The cell of any one of claims 28-50, wherein the cell further expresses a safety switch.
The cell of claim 51, wherein the safety switch is expressed on the cell surface and comprises one or more monoclonal antibody-specific epitopes.
The cell of any one of claims 51-52, wherein the safety switch comprises, in sequence: a CD8αsignal peptide, linker 1, a first monoclonal antibody-specific epitope, linker 2, a second monoclonal antibody-specific epitope, linker 3, a CD8 hinge region, and a CD8 transmembrane domain.
The cell of any one of claims 51-53, wherein the monoclonal antibody-specific epitopes are CD20 epitopes.
The cell of any one of claims 51-54, wherein the safety switch is co-expressed with the fusion protein comprising a first domain that activates an antigen-presenting cell (APC) and a second domain that activates an immune effector cell.
The cell of any one of claims 51-55, wherein the cell comprises an amino acid sequence as set forth in SEQ ID NO: 105 and/or a nucleotide sequence encoding the same.
The cell of any one of claims 28-56 that is an immune effector cell.
The cell of claim 57 that is derived from a cell isolated from peripheral blood or bone marrow.
The cell of claim 58 that is derived from a cell differentiated in vitro from a stem or progenitor cell selected from the group consisting of a T cell progenitor cell, a hematopoietic stem and progenitor cell, a hematopoietic multipotent progenitor cell, an embryonic stem cell, and an induced pluripotent cell.
The cell of any one of claims 28-59 that is a T cell or a NK cell.
The cell of claim 60 that is a cytotoxic T cell, a helper T cell, a gamma delta T, a CD4+/CD8+ double positive T cell, a CD4+ T cell, a CD8+ T cell, a CD4/CD8 double negative T cell, a CD3+ T cell, a naive T cell, an effector T cell, a helper T cell, a memory T cell, a regulator T cell, a Th0 cell, a Th1 cell, a Th2 cell, a Th3 (Treg) cell, a Th9 cell, a Th17 cell, a Thαβ helper cell, a Tfh cell, a stem memory TSCM cell, a central memory TCM cell, an effector memory TEM cell, or an effector memory TEMRA cell.
The cell of claim 60 that is a cytotoxic T cell.
A population of the cells comprising the cell of any one of claims 28-62, wherein the population of cells are derived from peripheral blood mononuclear cells (PBMC) , peripheral blood leukocytes (PBL) , tumor infiltrating lymphocytes (TIL) , cytokine-induced killer cells (CIK) , lymphokine-activated killer cells (LAK) , or marrow infiltrate lymphocytes (MILs) .
A pharmaceutical composition comprising a therapeutically effective amount of the antibody or antigen-binding fragment of any one of claims 1-9, the CAR of any one of claims 10-18, the polynucleotide of any one of claims 19-20, the vector of claim 21 and/or the cell or cell population of any one of claims 22 to 63, and a pharmaceutically acceptable carrier or excipient.
Use of the antibody or antigen-binding fragment of any one of claims 1-9, the CAR of any one of claims 10-18, the polynucleotide of any one of claims 19-20, the vector of claim 21, the cell or cell population of any one of claims 22-62 and/or the pharmaceutical composition of claim 64 for the preparation of a medicament for the treatment of cancer.
Use of the antibody or antigen-binding fragment of any one of claims 1-9, the CAR of any one of claims 10-18, the polynucleotide of any one of claims 19-20, the vector of claim 21, the cell or cell population of any one of claims 22-63 and/or the pharmaceutical composition of claim 64 in cancer treatment.
The use of claim 65 or 66, wherein the cell, population of cells, or pharmaceutical composition is used in combination with an additional therapy.
A method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment of any one of claims 1-9, the CAR of anyone of claims 10-18, the polynucleotide of any one of claims 19-20, the vector of claim 21, the cell of or cell population of any one of claims 22-63 and/or the pharmaceutical composition of claim 64.
The method of claim 68, wherein the cell or population of cells is autologous to the subject.
The method of claim 68, further comprising obtaining cells from the subject.
The method of any one of claims 68-70, further comprising administering an additional therapy to the subject.
The method of any one of claims 68-71, wherein the subject is a human.
The use of any one of claims 65-67 or the method of any one of claims 68-72, wherein the cancer is a HER2-expressing cancer.
The use of any one of claims 65-67 or the method of any one of claims 68-72, wherein the cancer is a solid tumor or a hematological cancer.
The use of any one of claims 65-67 or the method of any one of claims 68-72, wherein the cancer is breast cancer, gastric cancer, ovarian cancer, cervical cancer, urothelial cancer, esophageal cancer, bladder cancer, colorectal cancer, endometrial cancer, kidney cancer, lung cancer, pancreatic cancer, head and neck cancer, sarcoma, glioblastoma, prostate cancer, and/or thyroid cancer.
A method of preparing a cell capable of expressing a CAR that specifically binds HER2, comprising transferring the polynucleotide of claim 19 or 20 into the cell.
The method of claim 76, wherein the polynucleotide is transferred via electroporation.
The method of claim 76, wherein the polynucleotide is transferred via viral transduction.
The method of claim 78, comprising using a lentivirus, a retrovirus, an adenovirus, or an adeno-associated virus for the viral transduction.
The method of claim 76, wherein the polynucleotide is transferred using a transposon system.
The method of claim 80, wherein the transposon system is Sleeping Beauty or PiggyBac.
The method of claim 76, wherein the polynucleotide is transferred using gene-editing.
The method of claim 82, wherein the polynucleotide is transferred using a CRISPR-Cas system, a ZFN system, or a TALEN system.
The method of any one of claims 76-83, wherein the cell is selected from the group consisting of a T cell, an NK cell, an NKT cell, a macrophage, a neutrophil, and a granulocyte cell.