Human antibodies against FAP-alpha

Abstract

Further, fully human anti-FAPα antibodies and their variants with optimized CDR sequences are provided. These new antibodies have shown high affinity to FAPα protein and can be used to treat cancer and inflammatory conditions. According to one embodiment of the present disclosure, an antibody or antigen-binding fragment thereof is provided that has specificity to human fibroblast activation protein alpha (FAPα) protein, comprising a heavy chain variable region (VH) comprising VH CDR1, VH CDR2, and VH CDR3, and a light chain variable region (VL) comprising VL CDR1, VL CDR2, and VL CDR3.

Description

[Background technology]

[0001] background FAPα (fibroblast activation protein alpha, FAP, FAPA, or FAP alpha) is an integral membrane gelatinase that is overexpressed in cancer-associated fibroblasts (CAFs). CAFs are tumor stromal cells that represent the most prominent components of the tumor microenvironment. CAFs modulate multiple aspects of tumor growth, including tumorigenesis, angiogenesis, metastasis, immunosuppression, and promoting drug resistance. FAPα is thought to be involved in the control of fibroblast growth or epithelial-mesenchymal interactions during development, tissue repair, and epithelial carcinogenesis.

[0002] FAPα is selectively expressed in reactive stromal fibroblasts of epithelial cancers, granulation tissue of healing wounds, and malignant cells of bone and soft tissue sarcomas. Across a wide range of human cancer types, including gastric, pancreatic, breast, and colon cancers, expression of FAPα has been reported to correlate with higher tumor grade and poorer overall survival in solid tumors. Currently, FAP inhibitors are being widely developed in the field of tumor PET / computed tomography (CT) imaging. Theoretically, drugs targeting FAP may also serve as promising therapeutic targets for the inhibition of tumor progression and metastasis. However, no satisfactory therapeutic effects have been observed so far. Therefore, given the important role of FAPα in tumor progression and its rare expression in healthy tissues, it is worthwhile to further explore the therapeutic value of FAPα in the treatment of cancer.

[0003] In addition, it was observed that high expression of FAPα in RA (rheumatoid arthritis) fibroblast-like synoviocytes (FLS) was associated with an invasive phenotype of FLS accompanied by high proliferation and destruction of the extracellular matrix, and therefore FAPα can be considered as a therapeutic target for inhibiting the destructive power of FLS. Summary of the Invention [Means for solving the problem]

[0004] overview The present disclosure provides in various embodiments the fully human antibody and antigen-binding fragment specific to human FAPα protein.Experimental test shows that these newly identified antibodies can strongly and specifically bind to human FAPα protein.In addition, the majority of them retain strong affinity to FAPα protein expressed on cell surface, and can be suitably used to treat diseases related to FAPα expression.

[0005] According to one embodiment of the present disclosure, there is provided an antibody or antigen-binding fragment thereof having specificity for human fibroblast activation protein alpha (FAPα) protein, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) comprising VH CDR1, VH CDR2, and VH CDR3, and a light chain variable region (VL) comprising VL CDR1, VL CDR2, and VL CDR3.

[0006] In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 67; VH CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 15-17 and 43-55; VH CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-23, 25, 59 and 60; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 68; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 31; and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 69.

[0007] In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 67; VH CDR2 comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 15 and 43-48; VH CDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 20, 59 and 60; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 68; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 31; VL CDR3 comprises the amino acid sequence of SEQ ID NO: 69. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 11; VH CDR2 comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 15 and 43-48; VH CDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 20, 59 and 60; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 27; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 31; VL CDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 34, 61 and 62.

[0008] In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 67; VH CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 16 and 49-54; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 21; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 68; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 31; and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 69. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 12; VH CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 16 and 49-54; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 21; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 28; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 31; and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 35, 63, and 64.

[0009] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 67; the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 17 or 55; the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 22; the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 68; the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 31; and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 69. In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 12; the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 17 or 55; the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 22; the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 27; the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 31; and the VL CDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 34, 61 and 62.

[0010] In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 67; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 17 or 55; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 23; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 68; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 31; and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 69.

[0011] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 12; the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 17 or 55; the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 23; the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 27; the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 31; and the VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 36, 65, and 66.

[0012] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 67; the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 17 or 55; the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 25; the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 68; the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 31; and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 69. In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 12; the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 17 or 55; the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 25; the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 27; the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 31; and the VL CDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 34, 61 and 62.

[0013] Also provided are multispecific antibodies comprising an antigen-binding fragment of the disclosure and one or more antibodies or antigen-binding fragments that have binding specificity for a target antigen that is not FAPα.

[0014] In another embodiment, a chimeric antigen receptor (CAR) is also provided that comprises an antigen-binding fragment of the present disclosure, a transmembrane domain, a costimulatory domain, and a CD3ζ intracellular domain.

[0015] Polynucleotides encoding the antibodies or antigen-binding fragments thereof or CARs of the present disclosure are also provided. In some embodiments, the polynucleotide is an mRNA, optionally chemically modified.

[0016] Methods and uses for treating cancer and inflammatory conditions using the antibodies or antigen-binding fragments thereof of the present disclosure are also provided. [Brief description of the drawings]

[0017] [Figure 1] FIG. 1 shows the ELISA binding activity of all antibodies tested with human and mouse FAPα protein.

[0018] [Diagram 2] FIG. 2 shows that the antibodies tested, with the exception of I38 and J59, bound with high affinity to the FAPα protein on CHO-K1-hFAPα cells.

[0019] [Figure 3-1] FIG. 3 shows the results of the Biacore kinetic studies for each of the antibodies tested. [Figure 3-2] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] Detailed Description definition It should be noted that the term "a" or "an" entity refers to one or more of that entity, e.g., "an antibody" is understood to represent one or more antibodies. Thus, the terms "a" (or "an"), "one or more," and "at least one" may be used interchangeably herein.

[0021] As used herein, "antibody" or "antigen-binding fragment" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a whole antibody and any antigen-binding fragment or single chain. Thus, the term "antibody" includes any protein or peptide containing molecule that includes at least a portion of an immunoglobulin molecule that has the biological activity of binding to an antigen. Examples of such include, but are not limited to, the complementarity determining regions (CDRs) of a heavy or light chain or a ligand-binding portion thereof, a heavy or light chain variable region, a heavy or light chain constant region, a framework (FR) region, or any portion thereof, or at least a portion of a binding protein.

[0022] The term "antibody fragment" or "antigen-binding fragment" as used herein refers to a portion of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc. Regardless of structure, an antibody fragment binds with the same antigen recognized by the intact antibody. The term "antibody fragment" includes aptamers, spiegeleisen, and diabodies. The term "antibody fragment" also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex.

[0023] The term antibody encompasses a wide variety of classes of polypeptides that can be distinguished biochemically. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), with some subclasses within these (e.g., γ1-γ4). It is the nature of this chain that determines the "class" of the antibody, such as IgG, IgM, IgA, IgG, or IgE, respectively.

[0024] Immunoglobulin subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgG5, etc., are well characterized and known to provide functional specialization. Modified versions of each of these classes and isotypes are readily discernible to one of skill in the art in light of this disclosure and are therefore within the scope of this disclosure. All immunoglobulin classes are expressly within the scope of this disclosure, and the following discussion will generally refer to the IgG class of immunoglobulin molecules. With respect to IgG, a standard immunoglobulin molecule contains two identical light chain polypeptides of approximately 23,000 daltons molecular weight and two identical heavy chain polypeptides of 53,000-70,000 daltons molecular weight. The four chains are typically linked by disulfide bonds in a "Y" configuration, with the light chains flanking the heavy chains on either side, beginning at the mouth of the "Y" and continuing through the variable region.

[0025] Antibodies, antigen-binding polypeptides thereof, variants or derivatives of the disclosure include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primatized or chimeric antibodies, single chain antibodies, epitope-binding fragments such as Fab, Fab' and F(ab')2, Fd, Fv, single chain Fv (scFv), single chain antibodies, disulfide-linked Fv (sdFv), fragments comprising the VK or VH domains, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies to the antibodies disclosed herein). Immunoglobulin or antibody molecules of the disclosure can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule. Anti-FAPα antibody

[0026] As demonstrated in the attached experimental examples, the present inventors were able to generate fully human anti-FAPα antibodies G14, G52, I30, I37, I38, J40 and J59 (Table 1), all of which have high binding affinity to human FAPα protein. Furthermore, at least five of them, G14, G52, I30, I37 and J40, retained excellent affinity to cell surface FAPα, making them suitable molecules for therapeutic applications. Further experiments demonstrate that all of the antibodies target the same epitope on FAPα as the benchmark anti-FAPα antibody. Interestingly, these antibodies share highly homologous VH CDR1 and full VL CDR sequences.

[0027] According to one embodiment of the present disclosure, an antibody or its antigen-binding fragment is provided.In some embodiments, the antibody or its antigen-binding fragment has binding properties to human FAPα protein.In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) comprising VH CDR1, VH CDR2, and VH CDR3, and a light chain variable region (VL) comprising VL CDR1, VL CDR2, and VL CDR3.

[0028] In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 67; VH CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 15-17 and 39-47; VH CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-23, 25 and 51; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 31; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 68; and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 69.

[0029] As shown in Table 1C, SEQ ID NO:67 has a sequence of SYAMX, in which X is H or S. SEQ ID NO:68 has a sequence of RASQGX1X2SWLA, in which X1 is I or V and X2 is G or S. SEQ ID NO:69 has a sequence of QQAX1X2FPX3T, in which X1 is N or W, X2 is A or S and X3 is L, P or V.

[0030] In some embodiments, an antibody or antigen-binding fragment derived from antibody G14 is provided. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 67; VH CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 15 and 43-48; VH CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 20 and 51; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 68; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 31; and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 69.

[0031] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO:11, the VH CDR2 comprises the amino acid sequence of SEQ ID NO:15, the VH CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:20, the VL CDR1 comprises the amino acid sequence of SEQ ID NO:27, the VL CDR2 comprises the amino acid sequence of SEQ ID NO:31, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO:34.

[0032] In some embodiments, one or more of the CDRs are PTM (post-translational modification) risk avoided. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 11; VH CDR2 comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 15 and 43-48; VH CDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 20, 59 and 60; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 27; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 31; VL CDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 34, 61 and 62.

[0033] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 1 or comprises a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 1 and further retaining the VH CDR of SEQ ID NO: 1 or a PTM re-risked version thereof. In some embodiments, the VL comprises the amino acid sequence of SEQ ID NO: 2 or comprises a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 2 and further retaining the VL CDR of SEQ ID NO: 2 or a PTM re-risked version thereof. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 1 and the VL comprises the amino acid sequence of SEQ ID NO: 2.

[0034] Thus, in some embodiments, antibodies and antigen-binding fragments are provided that bind to the same epitope on FAPα as G14. Thus, in some embodiments, antibodies and antigen-binding fragments are provided that compete with G14 for binding to FAPα.

[0035] In some embodiments, an antibody or antigen-binding fragment derived from antibody G52 is provided. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 67; VH CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 16 and 49-54; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 21; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 68; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 31; and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 69.

[0036] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 12, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 16, and the VH CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 21, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 28, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 31, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 35.

[0037] In some embodiments, one or more of the CDRs are PTM (post-translational modification) risk avoided. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 12; VH CDR2 comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 16 and 49-54; VH CDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 21; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 28; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 31; VL CDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 35, 63 and 64.

[0038] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 3 or comprises a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 3 and further retaining the VH CDR of SEQ ID NO: 3 or a PTM re-risked version thereof. In some embodiments, the VL comprises the amino acid sequence of SEQ ID NO: 4 or comprises a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 4 and further retaining the VL CDR of SEQ ID NO: 4 or a PTM re-risked version thereof. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 3 and the VL comprises the amino acid sequence of SEQ ID NO: 4.

[0039] Thus, in some embodiments, antibodies and antigen-binding fragments are also provided that bind to the same epitope on FAPα as G52. Thus, in some embodiments, antibodies and antigen-binding fragments are also provided that compete with G52 for binding to FAPα.

[0040] In some embodiments, an antibody or antigen-binding fragment derived from antibody I30 is provided. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO:67; VH CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:17 and 55; VH CDR3 comprises the amino acid sequence of SEQ ID NO:22; VL CDR1 comprises the amino acid sequence of SEQ ID NO:68; VL CDR2 comprises the amino acid sequence of SEQ ID NO:31; and VL CDR3 comprises the amino acid sequence of SEQ ID NO:69.

[0041] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 12, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 17, and the VH CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 22, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 27, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 31, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 34.

[0042] In some embodiments, one or more of the CDRs are PTM (post-translational modification) risk avoided. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 12; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 17 or 55; VH CDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 22; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 27; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 31; VL CDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 34, 61 and 62.

[0043] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:5 or comprises a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO:5 and further retaining the VH CDR of SEQ ID NO:5 or a PTM re-risked version thereof. In some embodiments, the VL comprises the amino acid sequence of SEQ ID NO:6 or comprises a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO:6 and further retaining the VL CDR of SEQ ID NO:6 or a PTM re-risked version thereof. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:5 and the VL comprises the amino acid sequence of SEQ ID NO:6.

[0044] Thus, in some embodiments, antibodies and antigen-binding fragments are also provided that bind to the same epitope on FAPα as I30. Thus, in some embodiments, antibodies and antigen-binding fragments are also provided that compete with I30 for binding to FAPα.

[0045] In some embodiments, an antibody or antigen-binding fragment derived from antibody I37 is provided. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO:67; VH CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:17 and 55; VH CDR3 comprises the amino acid sequence of SEQ ID NO:23; VL CDR1 comprises the amino acid sequence of SEQ ID NO:68; VL CDR2 comprises the amino acid sequence of SEQ ID NO:31; VL CDR3 comprises the amino acid sequence of SEQ ID NO:69.

[0046] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 12, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 17, and the VH CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 23, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 27, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 31, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 36.

[0047] In some embodiments, one or more of the CDRs are PTM (post-translational modification) risk avoided. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 12; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 17 or 55; VH CDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 23; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 27; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 31; VL CDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 36, 65 and 66.

[0048] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:7 or comprises a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO:7 and further retaining the VH CDR of SEQ ID NO:7 or a PTM re-risked version thereof. In some embodiments, the VL comprises the amino acid sequence of SEQ ID NO:8 or comprises a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO:8 and further retaining the VL CDR of SEQ ID NO:8 or a PTM re-risked version thereof. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:7 and the VL comprises the amino acid sequence of SEQ ID NO:8.

[0049] Thus, in some embodiments, antibodies and antigen-binding fragments are also provided that bind to the same epitope on FAPα as I37. Thus, in some embodiments, antibodies and antigen-binding fragments are also provided that compete with I37 for binding to FAPα.

[0050] In some embodiments, an antibody or antigen-binding fragment derived from antibody J40 is provided. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO:67; VH CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:17 and 55; VH CDR3 comprises the amino acid sequence of SEQ ID NO:25; VL CDR1 comprises the amino acid sequence of SEQ ID NO:68; VL CDR2 comprises the amino acid sequence of SEQ ID NO:31; and VL CDR3 comprises the amino acid sequence of SEQ ID NO:69.

[0051] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 12, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 17, and the VH CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 25, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 27, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 31, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 34.

[0052] In some embodiments, one or more of the CDRs are PTM (post-translational modification) risk avoided. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 12; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 17 or 55; VH CDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 25; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 27; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 31; VL CDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 34, 61 and 62.

[0053] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:9 or comprises a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO:9 and further retaining the VH CDR of SEQ ID NO:9 or a PTM re-risked version thereof. In some embodiments, the VL comprises the amino acid sequence of SEQ ID NO:10 or comprises a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO:10 and further retaining the VL CDR of SEQ ID NO:10 or a PTM re-risked version thereof. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:9 and the VL comprises the amino acid sequence of SEQ ID NO:10.

[0054] Thus, in some embodiments, antibodies and antigen-binding fragments are also provided that bind to the same epitope on FAPα as J40. Thus, in some embodiments, antibodies and antigen-binding fragments are also provided that compete with J40 for binding to FAPα.

[0055] In some embodiments, an antibody or antigen-binding fragment is provided that is derived from antibody I38. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 13; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 18; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 24; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 29; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 32; VL CDR3 comprises the amino acid sequence of SEQ ID NO: 37.

[0056] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 39 or comprises a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 39 and further retaining the VH CDR of SEQ ID NO: 39 or a PTM re-risked version thereof. In some embodiments, the VL comprises the amino acid sequence of SEQ ID NO: 40 or comprises a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 40 and further retaining the VL CDR of SEQ ID NO: 40 or a PTM re-risked version thereof. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 39 and the VL comprises the amino acid sequence of SEQ ID NO: 40.

[0057] Thus, in some embodiments, antibodies and antigen-binding fragments are also provided that bind to the same epitope on FAPα as I38. Thus, in some embodiments, antibodies and antigen-binding fragments are also provided that compete with I38 for binding to FAPα.

[0058] In some embodiments, an antibody or antigen-binding fragment is provided that is derived from antibody J59. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 14; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 19; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 26; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 30; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 33; VL CDR3 comprises the amino acid sequence of SEQ ID NO: 38.

[0059] In some embodiments, one or more of the CDRs are PTM (post-translational modification) risk avoided. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 14; VH CDR2 comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 19 and 56-58; VH CDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 26; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 30; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 33; VL CDR3 comprises the amino acid sequence of SEQ ID NO: 38.

[0060] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 41 or comprises a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 41 and further retaining the VH CDRs of SEQ ID NO: 41 or a PTM re-risked version thereof. In some embodiments, the VL comprises the amino acid sequence of SEQ ID NO: 42 or comprises a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 42 and further retaining the VL CDRs of SEQ ID NO: 42 or a PTM re-risked version thereof. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 41 and the VL comprises the amino acid sequence of SEQ ID NO: 42.

[0061] Thus, in some embodiments, antibodies and antigen-binding fragments are provided that bind to the same epitope on FAPα as J59. Thus, in some embodiments, antibodies and antigen-binding fragments are provided that compete with J59 for binding to FAPα.

[0062] In some embodiments, antibodies and antigen-binding fragments are also provided that comprise CDR sequences derived from the CDR sequences of the disclosure with one, two or three amino acid substitutions, deletions and / or additions. multifunctional molecules

[0063] A multifunctional molecule comprising an antibody or antigen-binding fragment specific for FAPα, such as those disclosed herein, and one or more antibodies or antigen-binding fragments having specificity for a second antigen.

[0064] In some embodiments, the second antigen is a protein expressed on immune cells, such as T cells, B cells, monocytes, macrophages, neutrophils, dendritic cells, phagocytes, natural killer cells, eosinophils, basophils and mast cells.

[0065] In some embodiments, the second antigen is against CD3, CD47, PD1, PD-L1, LAG3, TIM3, CTLA4, VISTA, CSFR1, A2AR, CD73, CD39, CD40, CEA, HER2, CMET, 4-1BB, OX40, SIRPA, CD16, CD28, ICOS, CTLA4, BTLA, TIGIT, HVEM, CD27, VEGFR, or VEGF.

[0066] Different formats of bispecific antibodies are also provided. In some embodiments, each of the anti-FAP alpha fragment and the second fragment is independently selected from a Fab fragment, a single chain variable fragment (scFv), or a single domain antibody. In some embodiments, the bispecific antibody further comprises an Fc fragment.

[0067] Bifunctional molecules are also provided that do not simply comprise antibodies or antigen-binding fragments.As tumor antigen targeting molecules, antibodies or antigen-binding fragments specific for FAPα, such as those described herein, can be combined with immunocytokines or ligands, optionally via peptide linkers.Linked immunocytokines or ligands include, but are not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, GM-CSF, TNF-α, CD40L, OX40L, CD27L, CD30L, 4-1BBL, LIGHT and GITRL.Such bifunctional molecules can combine immune checkpoint blocking effect with tumor site local immune regulation. Chimeric Antigen Receptor

[0068] In one embodiment, a chimeric antigen receptor (CAR) is also provided, comprising the antibody or fragment thereof of the present disclosure as a targeting unit. In some embodiments, the CAR comprises the antibody or fragment thereof of the present disclosure, a transmembrane domain, a costimulatory domain, and a CD3ε intracellular domain.

[0069] The transmembrane domain can be designed to be fused to the extracellular domain, including antibody or fragment, optionally via hinge domain.It can also be fused to an intracellular domain, such as a costimulatory domain.In some embodiments, the transmembrane domain can include the natural transmembrane region of the costimulatory domain (e.g., the TM region of CD28T or 4-IBB used as costimulatory domain) or the natural transmembrane domain of the hinge region (e.g., the TM region of CD8alpha or CD28T used as hinge domain).

[0070] In some embodiments, a transmembrane domain may comprise a sequence that spans a cell membrane, but extends into the cytoplasm of the cell and / or into the extracellular space. For example, a transmembrane may comprise a membrane-spanning sequence that itself may further comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids that extend into the cytoplasm of the cell and / or into the extracellular space. Thus, a transmembrane domain may comprise a region that spans the membrane, and may further comprise amino acids that extend beyond the inner or outer surface of the membrane itself, and still be considered to be a "transmembrane domain".

[0071] In some embodiments, the transmembrane domain is fused to the cytoplasmic domain via a short linker. Optionally, a short peptide or polypeptide linker, preferably between 2 and 10 amino acids in length, can form the link between the transmembrane domain and the proximal cytoplasmic signaling domain of the chimeric receptor. A glycine-serine doublet (GS), a glycine-serine-glycine triplet (GSG), or an alanine-alanine-alanine triplet (AAA) would be a suitable linker.

[0072] In some embodiments, the CAR further comprises a costimulatory domain. In some embodiments, the costimulatory domain is located between the transmembrane domain and the activation domain. Examples of costimulatory domains include CD2, CD3 delta, CD3 epsilon, CD3 gamma, CD4, CD7, CD8a, CD8, CD11a (ITGAL), CD11b (ITGAM), CD11c (ITGAX), CD11d (ITGAD), CD18 (ITGB2), CD19 (B4), CD27 (T FRSF7), CD28, CD28T, CD29(ITGB1), CD30(TNFRSF8), CD40(TNFRSF5), CD48(SLAMF2), CD49a(ITGA1), CD49d(ITGA4), CD49f(ITGA6), CD66a(CEACAM1), CD66b(CEACAM8), CD66c(CEACAM6), CD66d(CEACAM3), CD66e(CEACAM5), CD69(CLEC2), CD79A(B cell antigen receptor complex-associated alpha chain), CD79B(B cell antigen receptor complex-associated beta chain), CD84(SLAMF5), CD96(Tactile), CD100(SEMA4D), CD103 (ITGAE), CD134(OX40), CD137(4-1BB), CD150(SLAMF1), CD158A(KIR2DL1), CD158B1(KI R2DL2), CD158B2(KIR2DL3), CD158C(KIR3DP1), CD158D(KIRDL4), CD158F1(KIR2DL5A), C D158F2(KIR2DL5B), CD158K(KTR3DL2), CD160(BY55), CD162(SELPLG), CD226(DNAM1), CD 229(SLAMF3), CD244(SLAMF4), CD247(CD3-zeta), CD258(LIGHT), CD268(BAFFR), CD270(T FSF14), CD272(BTLA), CD276(B7-H3), CD279(PD-1), CD314(KG2D), CD319(SLAMF7), CD335(K-p46 ), CD336(K-p44), CD337(K-p30), CD352(SLAMF6), CD353(SLAMF8), CD355(CRTAM), CD357(TNFRSF 18), inducible T cell costimulatory factor (ICOS), LFA-1 (CD 11a / CD 18), KG2C, DAP-10, ICAM-1, Kp80 (KLRF1), IL-2R beta, IL-2R gamma, IL-7R alpha, LFA-1, SLAMF9, LAT, GADS (GrpL), SLP-76 (LCP2), PAG1 / CBP, CD83 ligand, Fc gamma receptor, MHC class 1 molecule, MHC class 2 molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, activating NK cell receptor, Toll ligand receptor, and fragments or combinations thereof.

[0073] In some embodiments, the cytoplasmic portion of the CAR also comprises a signaling / activation domain. In one embodiment, the signaling / activation domain is a CD3 zeta domain or an amino acid sequence having at least about 80%, 85%, 90%, 95%, 98% or 99% sequence identity to a CD3 zeta domain. Methods for expressing or preparing polynucleotides, mRNA, and antibodies

[0074] The present disclosure also provides a polynucleotide or nucleic acid molecule that encodes the antibody, its variant or derivative, or CAR of the present disclosure.The polynucleotide of the present disclosure can encode the entire heavy and light chain variable region of the antigen-binding polypeptide, its variant or derivative on the same polynucleotide molecule or on separate polynucleotide molecules.In addition, the polynucleotide of the present disclosure can encode the part of the heavy and light chain variable region of the antigen-binding polypeptide, its variant or derivative on the same polynucleotide molecule or on separate polynucleotide molecules.

[0075] In some embodiments, the polynucleotide is an mRNA molecule. In some embodiments, the mRNA can be introduced into a target cell for expression of the antibody or fragment thereof.

[0076] mRNA can be synthesized according to any of a variety of known methods. For example, mRNA can be synthesized by in vitro transcription (IVT). Briefly, IVT is typically carried out using linear or circular template DNA, containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may contain DTT and magnesium ions, and a suitable RNA polymerase (e.g., T3, T7 or SP6 RNA polymerase), DNAse I, pyrophosphatase, and / or RNAse inhibitor. Stringent conditions vary depending on specific applications.

[0077] In some embodiments, for the preparation of mRNA encoding an antibody, the template DNA is transcribed in vitro. A suitable template DNA typically has a promoter for in vitro transcription, such as a T3, T7 or SP6 promoter, followed by the desired nucleotide sequence for the mRNA encoding the desired antibody (e.g., encoding a heavy or light chain), and a termination signal.

[0078] Using standard methods, the mRNA sequence encoding the desired antibody (e.g., encoding a heavy or light chain) can be determined and incorporated into the template DNA. For example, starting from the desired amino acid sequence (e.g., the desired heavy or light chain sequence), a virtual back-translation is performed based on the degenerate genetic code. An optimization algorithm can then be used to select the appropriate codons. Typically, the G / C content can be optimized on the one hand to achieve as high a G / C content as possible, and on the other hand to take into account as much as possible the frequency of tRNA according to the codon usage frequency. The optimized RNA sequence can be established and displayed, for example with the aid of a suitable display device, and compared with the original (wild type) sequence. The secondary structure can also be analyzed to calculate the stabilizing and destabilizing properties or, respectively, regions of the RNA.

[0079] mRNA can be synthesized as unmodified or modified mRNA.Typically, mRNA is modified to improve stability.Modification of mRNA can include, for example, modification of nucleotide of RNA.Thus, modified mRNA can include, for example, backbone modification, sugar modification or base modification.In some embodiments, the mRNA encoding the antibody (e.g., mRNA encoding the heavy and light chains) can be synthesized from naturally occurring nucleotides and / or nucleotide analogs (modified nucleotides), including purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil)), as well as modified nucleotides such as purines and pyrimidine analogs. Analogs or derivatives, such as 1-methyl-adenine, 2-methyl-adenine, 2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine, N6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl-cytosine, 4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine, 1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine, 7-methyl-guanine, inosine, 1-methyl-inosine, pseudouracil (5-uracil), uracil), dihydro-uracil, 2-thio-uracil, 4-thio-uracil, 5-carboxymethylaminomethyl-2-thio-uracil, 5-(carboxyhydroxymethyl)-uracil, 5-fluoro-uracil, 5-bromo-uracil, 5-carboxymethylaminomethyl-uracil, 5-methyl-2-thio-uracil, 5-methyl-uracil, N-uracil-5-oxyacetic acid methyl ester, 5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thio-uracil, These include, but are not limited to, 5'-methoxycarbonylmethyl-uracil, 5-methoxy-uracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 1-methyl-pseudouracil, queosine, 13-D-mannosyl-queosine, wybutoxosine, as well as phosphoramidates, phosphorothioates, peptide nucleotides, methylphosphonates, 7-deazaguanosine, 5-methylcytosine and inosine.The preparation of such analogs is known to those skilled in the art, for example from U.S. Pat. Nos. 4,373,071, 4,401,796, 4,415,732, 4,458,066, 4,500,707, 4,668,777, 4,973,679, 5,047,524, 5,132,418, 5,153,319, 5,262,530 and 5,700,642, the disclosures of which are incorporated herein by reference in their entirety.

[0080] In some embodiments, mRNA (e.g., mRNA encoding heavy and light chains) may contain RNA backbone modification. Typically, backbone modification is a modification in which the backbone phosphate of the nucleotide contained in RNA is chemically modified. Exemplary backbone modifications typically include, but are not limited to, modifications from the group consisting of methylphosphonate, methylphosphoramidate, phosphoramidate, phosphorothioate (e.g., cytidine 5'-O-(1-thiophosphate)), boranophosphate, positively charged guanidinium, etc., which means that phosphodiester bond is replaced by other anionic, cationic or neutral groups.

[0081] In some embodiments, the mRNA (e.g., mRNA encoding the heavy and light chains) may contain sugar modifications. Exemplary sugar modifications are chemical modifications of the sugars of the nucleotides they contain, such as 2'-deoxy-2'-fluoro-oligoribonucleotides (2'-fluoro-2'-deoxycytidine 5'-triphosphate, 2'-fluoro-2'-deoxyuridine 5'-triphosphate), 2'-deoxy-2'-deamine-oligoribonucleotides (2'-amino-2'-deoxycytidine 5'-triphosphate, 2'-amino-2'-deoxyuridine 5'-triphosphate), 2'-O-alkyloligoribonucleotides, 2'-deoxy-2'-fluoro-2'-deoxyuridine 5'-triphosphate, 2'-fluoro-2'-deoxyuridine 5'-triphosphate, 2'-amino ... and 2'-C-alkyl oligoribonucleotides, and their isomers (2'-aracytidine 5'-triphosphate, 2'-aruridine 5'-triphosphate), or azido triphosphates (2'-azido-2'-deoxycytidine 5'-triphosphate, 2'-azido-2'-deoxyuridine 5'-triphosphate).

[0082] In some embodiments, the mRNA (e.g., mRNA encoding the heavy and light chains) may contain modifications of the base of a nucleotide (base modification). Modified nucleotides containing base modifications are also referred to as base-modified nucleotides. Examples of such base-modified nucleotides include 2-amino-6-chloropurine riboside 5'-triphosphate, 2-aminoadenosine 5'-triphosphate, 2-thiocytidine 5'-triphosphate, 2-thiouridine 5'-triphosphate, 4-thiouridine 5'-triphosphate, 5-aminoallylcytidine 5'-triphosphate, 5-aminoallyluridine 5'-triphosphate, 5-bromocytidine 5'-triphosphate, 5-bromouridine 5'-triphosphate, 5-iodocytidine 5'-triphosphate, 5-iodouridine 5'-triphosphate, 5-methylcytidine 5'-triphosphate, 5-methyluridine 5'-triphosphate, and 6-azacytidine 5'-triphosphate. , 6-azauridine 5'-triphosphate, 6-chloropurine riboside 5'-triphosphate, 7-deazaadenosine 5'-triphosphate, 7-deazaguanosine 5'-triphosphate, 8-azaadenosine 5'-triphosphate, 8-azidoadenosine 5'-triphosphate, benzimidazole riboside 5'-triphosphate, N1-methyladenosine 5'-triphosphate, N1-methylguanosine 5'-triphosphate, N6-methyladenosine 5'-triphosphate, O6-methylguanosine 5'-triphosphate, pseudouridine 5'-triphosphate, puromycin 5'-triphosphate or xanthosine 5'-triphosphate.

[0083] Typically, mRNA synthesis involves the addition of a "cap" to the N-terminal (5') end and a "tail" to the C-terminal (3') end. The presence of the cap is important in making the mRNA resistant to nucleases found in most eukaryotic cells. The presence of the "tail" helps protect the mRNA from exonuclease degradation.

[0084] Thus, in some embodiments, the mRNA (e.g., mRNA encoding the heavy and light chains) includes a 5' cap structure. The 5' cap is typically added as follows: first, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5' nucleotide, leaving two terminal phosphates; then, guanosine triphosphate (GTP) is added to the terminal phosphate by guanylyltransferase, resulting in a 5'5'5 triphosphate linkage; then, the 7-nitrogen of guanine is methylated by a methyltransferase. Examples of cap structures include, but are not limited to, m7G(5')ppp(5'(A,G(5')ppp(5)A and G(5)ppp(5')G.

[0085] In some embodiments, the mRNA (e.g., mRNA encoding the heavy and light chains) comprises a 3' poly(A) tail structure. The polyA tail at the 3' end of the mRNA typically comprises about 10-300 adenosine nucleotides (e.g., about 10-200 adenosine nucleotides, about 10-175 adenosine nucleotides, about 10-150 adenosine nucleotides, about 10-125 adenosine nucleotides, 10-100 adenosine nucleotides, about 10-75 adenosine nucleotides, about 20-70 adenosine nucleotides, or about 20-60 adenosine nucleotides). In some embodiments, the mRNA encoding the antibody (e.g., mRNA encoding the heavy and light chains) comprises a 3' poly(C) tail structure. A suitable poly-C tail at the 3' end of an mRNA typically comprises about 10-200 cytosine nucleotides (e.g., about 10-150 cytosine nucleotides, about 10-100 cytosine nucleotides, about 20-70 cytosine nucleotides, about 20-60 cytosine nucleotides, or about 10-40 cytosine nucleotides). The poly-C tail can be added to or replace the poly-A tail.

[0086] In some embodiments, the mRNA (e.g., mRNAs encoding the heavy and light chains) comprises a 5' and / or 3' untranslated region. In some embodiments, the 5' untranslated region comprises one or more elements that affect mRNA stability or translation, e.g., an iron response element. In some embodiments, the 5' untranslated region can be between about 50 and 500 nucleotides in length (e.g., between about 50 and 400 nucleotides in length, between about 50 and 300 nucleotides in length, between about 50 and 200 nucleotides in length, or between about 50 and 100 nucleotides in length).

[0087] In some embodiments, the 5' region of the mRNA (e.g., mRNA encoding the heavy and light chains) comprises a sequence encoding a signal peptide, such as those described herein. In certain embodiments, a signal peptide derived from human growth hormone (hGH) is incorporated into the 5' region. Typically, the sequence encoding the signal peptide is linked directly or indirectly to the sequence encoding the heavy or light chain at the N-terminus.

[0088] This technology can be used to deliver any antibody known in the art and that can be raised against a desired antigen using standard methods. The invention can be used to deliver monoclonal antibodies, polyclonal antibodies, antibody mixtures or cocktails, human or humanized antibodies, chimeric antibodies, or bispecific antibodies.

[0089] Methods for making antibodies are well known in the art and are described herein. In certain embodiments, both the variable and constant regions of the antigen-binding polypeptide of the present disclosure are fully human. Fully human antibodies can be made using techniques described in the art and as described herein. For example, fully human antibodies against a specific antigen can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigen administration but has an inactive endogenous locus. Exemplary techniques that can be used to make such antibodies are described in U.S. Patent Nos. 6,150,584, 6,458,592, and 6,420,140, ​​which are incorporated herein by reference in their entirety. Treatment and Use

[0090] As described herein, the antibodies, variants, derivatives, or antibody-drug conjugates of the disclosure can be used in certain treatment and diagnostic methods.

[0091] The present disclosure further relates to antibody-based therapy, including administering the disclosed antibodies, fragments, or antibody-drug conjugates to patients, such as animals, mammals, and humans, to treat one or more of the disorders or conditions described herein. Therapeutic compounds of the present disclosure include, but are not limited to, the disclosed antibodies (including variants and derivatives thereof as described herein) and nucleic acids or polynucleotides encoding the disclosed antibodies (including variants and derivatives thereof as described herein).

[0092] The antibody of the present disclosure can also be used to treat or inhibit cancer.As provided above, FAPα can be overexpressed in tumor cells, particularly in liver, stomach, pancreas, esophagus, ovary and lung tumors.Inhibition of FAPα has been shown to be useful in tumor treatment.

[0093] Thus, in some embodiments, a method for treating cancer in a patient in need thereof is provided. The method, in one embodiment, entails administering to the patient an effective amount of the antibody, fragment, or antibody-drug conjugate of the present disclosure. In some embodiments, at least one of the cancer cells (e.g., stromal cells) in the patient overexpresses FAPα.

[0094] Cell therapy, for example, chimeric antigen receptor (CAR) T cell therapy, is also provided in the present disclosure. Suitable cells can be used that are transduced with a vector that encodes or contacts a CAR that includes (or alternatively is engineered to express) the anti-FAPα antibody of the present disclosure. Thus, by such contact or manipulation, the cells can be introduced into a cancer patient that requires treatment. The cancer patient can have any type of cancer as disclosed herein. The cells (e.g., T cells) can be, for example, but are not limited to, tumor-infiltrating T lymphocytes, CD4+ T cells, CD8+ T cells, or combinations thereof.

[0095] In some embodiments, the cells are isolated from the cancer patient himself or herself. In some embodiments, the cells are provided by a donor or from a cell bank. If the cells are isolated from the cancer patient, unwanted immune responses can be minimized.

[0096] Non-limiting examples of cancer include bladder cancer, breast cancer, colon cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, pancreatic cancer, prostate cancer, and thyroid cancer. In some embodiments, the cancer is one or more of gastric cancer, pancreatic cancer, esophageal cancer, ovarian cancer, and lung cancer.

[0097] Additional diseases or conditions associated with increased cell survival that may be treated, prevented, diagnosed and / or prognosed by the disclosed antibodies or variants, or derivatives thereof, include, but are not limited to, progression and / or metastasis of malignancies and related disorders, such as leukemias (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's hypergammaglobulinemia, heavy chain disease, and solid tumors, including, but not limited to, sarcomas and Carcinomas, e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovium, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, These include, but are not limited to, alveolar carcinoma, hepatoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0098] FAPα is involved in the regulation of fibroblast growth or epithelial-mesenchymal interactions during development, tissue repair and epithelial carcinogenesis. High expression in RA (rheumatoid arthritis) fibroblast-like synoviocytes (FLS) was observed to be associated with an invasive phenotype of FLS accompanied by high proliferation and destruction of the extracellular matrix. Therefore, FAPα can be considered as a therapeutic target for inhibiting the destructive power of FLS.

[0099] Methods and uses for treating inflammatory conditions or autoimmune diseases are also provided. In some embodiments, the inflammatory diseases or conditions treated by the disclosed antibodies, fragments and compositions include one or more of Alzheimer's disease, Addison's disease, atherosclerosis, ankylosing spondylitis, arthritis, osteoarthritis (OA), rheumatoid arthritis (RA), psoriatic arthritis (PA), ankylosing spondylitis, asthma, atherosclerosis, chronic obstructive pulmonary disease (COPD), Crohn's disease, colitis, dermatitis, interstitial inflammation, fibromyalgia, hepatitis, irritable bowel syndrome (IBS), systemic lupus erythematous (SLE), nephritis, Parkinson's disease (PD), vasculitis, and ulcerative colitis.

[0100] In some embodiments, the autoimmune disease or condition treated by the disclosed antibodies, fragments, and compositions comprises one or more of alopecia areata, autoimmune hemolytic anemia, autoimmune hepatitis, dermatomyositis, diabetes mellitus (type 1), celiac disease, autoimmune juvenile idiopathic arthritis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, idiopathic thrombocytopenic purpura, gravitational myasthenia, autoimmune myocarditis, multiple sclerosis, pemphigus / pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma / systemic sclerosis, Sjogren's syndrome, systemic lupus erythematosus, autoimmune thyroiditis, Hashimoto's thyroiditis, autoimmune uveitis, vitiligo, and granulomatosis with polyangiitis (Wegener's granulomatosis).

[0101] Rheumatoid arthritis (RA) is a long-term autoimmune disorder that primarily affects the joints. As a result, the joints typically become hot, swollen, and painful. The pain and stiffness often worsen after rest. The wrists and hands are usually affected, and the same joints on both sides of the body are typically affected. The disease can also affect other parts of the body. The cause of rheumatoid arthritis is unknown, but it is thought to involve a combination of genetic and environmental factors. The underlying mechanism involves the body's immune system attacking the joints. This results in inflammation and thickening of the joint capsule. The goal of treatment is to relieve pain, reduce inflammation, and improve a person's overall function. Painkillers, steroids, and NSAIDs are frequently used to help with symptoms. A group of drugs called disease-modifying antirheumatic drugs (DMARDs), such as hydroxychloroquine and methotrexate, may be used in an attempt to slow the progression of the disease.

[0102] The specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including the specific antibody, its variant or derivative used, the patient's age, weight, general health, sex and diet, as well as the number of administrations, excretion rate, drug combinations, and the severity of the particular disease being treated.The judgment of such factors by medical practitioners is within the ordinary skill of the art.The amount will also depend on the individual patient to be treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease, and the desired effect.The amount used can be determined by the principles of pharmacology and pharmacokinetics well known in the art.

[0103] Methods of administration of antibodies, fragments, or antibody-drug conjugates include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. Antigen-binding polypeptides or compositions can be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can be administered together with other bioactive agents. Thus, pharmaceutical compositions containing antigen-binding polypeptides of the present disclosure can be administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as powders, ointments, drops, or transdermal patches), bucally, or as an oral spray or transdermal patch. It can be administered as a nasal spray.

[0104] The term "parenteral" as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

[0105] Administration can be systemic or local.In addition, it may be desirable to introduce the antibody of the present disclosure into the central nervous system by any suitable route, including intraventricular and intrathecal injection, and intraventricular injection can be facilitated by an intraventricular catheter, for example, attached to a reservoir such as an Ommaya reservoir.Pulmonary administration can also be utilized, for example, by using an inhaler or nebulizer, and a formulation containing an aerosolizing agent.

[0106] It may be desirable to administer an antigen-binding polypeptide or composition of the disclosure locally to the area in need of treatment, which can be achieved by, for example and not by way of limitation, local infusion during surgery, topical application, e.g., in conjunction with wound dressing after surgery, by injection, by catheter, by suppository, or by a deposit, said deposit being of a porous, nonporous or gelatinous material, including membranes, e.g., sialastic membranes, or fibers. Preferably, when administering proteins, including antibodies, of the disclosure, care should be taken to use materials to which the proteins do not absorb.

[0107] The amount of the antibody, fragment, or antibody-drug conjugate of the present disclosure that will be effective in treating, inhibiting, and preventing inflammatory, immune, or malignant diseases, disorders, or conditions can be determined by standard clinical techniques. In addition, in vitro assays can be used as necessary to help identify optimal dosage ranges. The exact dose to be employed in the formulation will also depend on the route of administration and the severity of the disease, disorder, or condition, and should be determined according to the judgment of the practitioner and each patient's circumstances. An effective dose can be determined by in vitro assays. Dose-response curves derived from in vitro or animal model test systems can be extrapolated.

[0108] As a general proposition, the dosage of the antibody, fragment, or antibody-drug conjugate of the present disclosure administered to a patient is typically between 0.001 mg and 100 mg per kg of the patient's body weight, between 0.01 mg and 20 mg per kg of the patient's body weight, or between 0.5 mg and 10 mg per kg of the patient's body weight. Generally, human antibodies have a longer half-life in the human body than antibodies from other animal species due to the immune response to the foreign polypeptides. Thus, lower dosages and less frequent administration of human antibodies are often possible. Additionally, the dosage and frequency of administration of the antibodies of the present disclosure can be reduced by enhancing antibody uptake and tissue penetration (e.g., into the brain) by modifications such as lipidation.

[0109] In additional embodiments, the compositions of the present disclosure are administered in combination with cytokines. Cytokines that may be administered with the compositions of the present disclosure include, but are not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, anti-CD40, CD40L, and TNF-α.

[0110] In additional embodiments, the compositions of the present disclosure are administered in combination with other therapeutic or prophylactic regimens, such as, for example, radiation therapy. composition

[0111] The present disclosure also provides pharmaceutical compositions. Such compositions comprise an effective amount of antibody, fragment, or antibody-drug conjugate and acceptable carrier. In some embodiments, the composition further comprises a second anti-cancer agent (e.g., immune checkpoint inhibitor).

[0112] In specific embodiments, the term "pharmaceutically acceptable" means approved by a regulatory agency of a federal or state government, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias, for use in animals, and more particularly in humans. Moreover, a "pharmaceutically acceptable carrier" will generally be a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation aid of any type.

[0113] The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be utilized as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, glycol, water, ethanol, and the like. The composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, such as acetates, citrates, or phosphates, if desired. Antibacterial agents, such as benzyl alcohol or methyl parabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; and agents for adjusting osmotic tonicity, such as sodium chloride or dextrose, are also contemplated. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, and the like. The compositions can be formulated as suppositories using traditional binders and carriers, such as triglycerides. Oral formulations can include standard carriers, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by EW Martin, which is incorporated herein by reference. Such compositions will contain a therapeutically effective amount of the antigen-binding polypeptide, preferably in purified form, together with a suitable amount of carrier to provide the form for proper administration to the patient. The formulation must be suitable for the method of administration. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0114] In an embodiment, the composition is formulated according to routine procedures as a pharmaceutical composition adapted for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Optionally, the composition may also include a solubilizing agent and a local anesthetic, such as lignocaine, to ease pain at the injection site. Generally, these ingredients are supplied either separately or mixed in unit dosage form, for example as a dry frozen powder or water-free concentrate in a hermetically sealed container, such as an ampoule or sachet indicating the amount of active agent. If the composition is to be administered by injection, it can be dispensed using an injection bottle containing sterile water or saline of pharmaceutical grade. If the composition is to be administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0115] The compounds of the present disclosure can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc. EXAMPLES

[0116] Example 1 Fully human naive phage library panning and screening This example screened for fully human anti-FAPα antibodies from a human naive phage library.

[0117] Antigen: human FAPα-His tag (Sino biological).

[0118] Preparation of a fully human naive phage library: A phage library was constructed by using a phagemid vector consisting of antibody gene fragments amplified from PBMCs of healthy human subjects. The library format is a Fab phage library. The library size is 5.2×10 10 It was.

[0119] Phage library solid-phase and solution panning against human FAPα protein. For solution panning, the phage library was first negatively screened by incubation with BSA-coated streptavidin Dynabeads. The resulting phages were incubated with biotinylated FAPα-His tagged protein and washed with Kingfihser magnetic bead system. Binders were eluted with trypsin. For solid-phase panning, the phage library was first negatively blocked with 5% PBSM. The resulting phages were incubated with FAPα-His tagged protein and washed with PBST+PBS. Binders were eluted with trypsin.

[0120] Solid-phase panning was combined with solution panning to comprehensively screen potential binders. The eluted phages were then tested for their titer of binding to the antigen and co-cultured with E. coli. There were five rounds of panning and screening. The titers of output 4 and output 5 were significantly increased.

[0121] Single clones were carefully selected from output 4 and 5, then cultured in 96-deep well plates. Culture supernatants were subjected to IgG enrichment and antigen binding titer evaluation. A total of 390 positive clones were selected and subjected to sequencing. After sequence analysis, 176 unique sequences were identified. All of these clones were subjected to ELISA binding analysis. The top 7 sequences were identified. See the table below. The sequences are provided in Table 1A below. Table 1A. Candidate antibody sequences [Table 1A-1] [Table 1A-2]

[0122] The CDR sequences of these antibodies (according to the Kabat system) are listed below in Table 1B. Also shown are variants thereof in which potential sites of post-translational modification (PTM) have been mutated to biologically equivalent ones (e.g., NG→NA, NG→QG, NS→NA, NS→QS, DG→DA, and DS→DA). These substitutions are envisioned to retain the biological activity of the antibodies while preventing PTM for ease of manufacturing. Table 1B. CDR sequences [Table 1B-1] [Table 1B-2]

[0123] Some of these antibodies, particularly G14, G52, I30, I37 and J40, have highly homologous VH CDR1 and VL CDR sequences. These CDR sequences are assumed to be interchangeable. Their alignment and consensus sequences are shown in Table 1C. Table 1C. Consensus CDRs [Table 1C-1] [Table 1C-2]

[0124] Example 2 Binding activity to human and mouse FAPα antigen This example tested the binding activity of antibodies against human and mouse FAPα proteins. 2.1 ELISA binding to human FAPα

[0125] To evaluate the binding activity of the clones, the tested antibodies were subjected to an ELISA test.

[0126] Briefly, microtiter plates were coated with 100 μl / well of human FAPα-His protein at 0.5 ug / ml in PBS overnight at 4°C, then blocked with 150 μl / well of 1% BSA. Three-fold dilutions of tested antibodies starting at 1 ug / ml were added to each well and incubated for 1 hour at 37°C. Plates were washed with PBS / Tween® and then incubated with anti-human IgG (H&L) (goat) antibody-peroxidase conjugate for 30 minutes at 37°C. After washing, plates were developed with TMB substrate and analyzed by spectrophotometer at OD 450 nm. As shown in Figure 1A and Table 2, G14, G52, I30, I37, I38, J40 and J59 clones all bound human FAPα with high affinity. The FAP-4B9 clone from Roche served as a positive control and was designated FAPα-BMK. 2.1 ELISA binding to mouse FAPα

[0127] To evaluate the binding activity of the clones, the tested antibodies were subjected to an ELISA test.

[0128] Briefly, microtiter plates were coated with 100 μl / well of mouse FAPα-His protein at 0.5 ug / ml in PBS overnight at 4°C, then blocked with 150 μl / well of 1% BSA. Three-fold dilutions of tested antibodies starting at 1 ug / ml were added to each well and incubated for 1 hour at 37°C. Plates were washed with PBS / Tween® and then incubated with anti-human IgG (H&L) (goat) antibody-peroxidase conjugate for 30 minutes at 37°C. After washing, plates were developed with TMB substrate and analyzed by spectrophotometer at OD 450 nm. As shown in Figure 1B and Table 2, only I37 and FAPα-BMK bound with high affinity to mouse FAPα. Table 2. Binding activity to human and mouse FAPα [Table 2] 2.2 Cell-based binding to human FAPα

[0129] To assess cell-based binding properties to human FAPα, the tested antibodies were analyzed by FACS for their binding to CHO-K1-hFAPα. A total of 1×10 cells were collected in each well. 5 CHO-K1-hFAPα cells were incubated with 4-fold serial dilutions of antibodies starting from 3 μg / ml in FACS buffer for 30 min at 4°C. After washing with FACS buffer, PE-conjugated anti-human IgG antibodies were added to each well and incubated for 30 min at 4°C. After washing, the MFI of PE was evaluated by MACSQuant Analyzer 16. As shown in Figure 2 and Table 3, G14, I30, I37 and J40 antibodies showed binding ability comparable to the positive reference "FAPα BMK antibody", while I38 and J59 clones dramatically lost binding activity. Table 3. Binding activity to CHO-K1-hFAPα cells [Table 3] 2.3 Protein dynamics for human FAPα

[0130] Binding of the tested antibodies to recombinant FAPα protein (human FAPα-his tag) was tested on Biacore using the capture method. mAbs were captured using a Protein A chip. Serial dilutions of human FAPα-his tag protein were injected over the captured antibody for 3 minutes at a flow rate of 30 μl / min. Antigen was allowed to dissociate for 800 seconds. All experiments were performed on a Biacore T200. Data analysis was performed using the Biacore T200 evaluation software. The results are shown in Figure 3 and Table 4 below. Table 4: Total kinetics measured by Biacore [Table 4]

[0131] Example 3 Epitope binning by competitive ELISA Competitive ELISA was performed to classify FAPα mAbs based on their binding epitopes on human FAPα.

[0132] Briefly, microtiter plates were coated with 0.5 μg / ml human FAPα protein in PBS, 100 μl / well, overnight at 4° C., and then blocked with 150 μl / well 1% BSA. Serial dilutions of chimeric antibodies and biotin-conjugated reference mAbs were added to each well and incubated for 1 h at RT. Plates were washed with PBS / Tween® and then incubated with streptavidin-HRP for 15 min at RT. After washing, plates were developed with TMB substrate and analyzed by spectrophotometer at OD 450 nm. Depending on their ability to compete with the reference mAb (Benchmark, or "BMK"), all tested FAPα mAbs bind to similar epitopes, as shown in Table 5. Table 5: Epitope binning study by ELISA. [Table 5] * * *

[0133] The present disclosure is not limited in scope by the specific embodiments described, which are intended as single illustrations of individual aspects of the present disclosure, and any compositions or methods that are functionally equivalent are within the scope of the present disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made to the methods and compositions of the present disclosure without departing from the spirit and scope of the present disclosure. Thus, the present disclosure encompasses modifications and variations of the present disclosure, provided that they fall within the scope of the appended claims and their equivalents.

[0134] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. An antibody or antigen-binding fragment thereof having specificity for human fibroblast activation protein alpha (FAPα) protein, comprising a heavy chain variable region (VH) comprising a VH CDR1, a VH CDR2, and a VH CDR3, and a light chain variable region (VL) comprising a VL CDR1, a VL CDR2, and a VL CDR3; the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 67; the VH CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 15-17 and 43-55; the VH CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-23, 25, 59, and 60; the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 68; the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 31; the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 69; An antibody or an antigen-binding fragment thereof.

2. the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 67; the VH CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 15 and 43-48; the VH CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 59, and 60; the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 68; the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 31; the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 69; The antibody or antigen-binding fragment thereof according to claim 1.

3. the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 11; the VH CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 15 and 43-48; the VH CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 59, and 60; the VL CDR1 comprises the amino acid sequence of SEQ ID NO:27; the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 31; the VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 34, 61 and 62; The antibody or antigen-binding fragment thereof according to claim 1.

4. The antibody or antigen-binding fragment thereof according to claim 3, wherein the VH comprises the amino acid sequence of SEQ ID NO: 1 and the VL comprises the amino acid sequence of SEQ ID NO:

2.

5. the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 67; the VH CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 16 and 49-54; the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 21; the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 68; the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 31; the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 69; The antibody or antigen-binding fragment thereof according to claim 1.

6. the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 12; the VH CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 16 and 49-54; the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 21; the VL CDR1 comprises the amino acid sequence of SEQ ID NO:28; the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 31; the VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 35, 63 and 64; The antibody or antigen-binding fragment thereof according to claim 5.

7. The antibody or antigen-binding fragment thereof according to claim 6, wherein the VH comprises the amino acid sequence of SEQ ID NO: 3 and the VL comprises the amino acid sequence of SEQ ID NO:

4.

8. the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 67; the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 17 or 55; the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 22; the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 68; the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 31; the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 69; The antibody or antigen-binding fragment thereof according to claim 1.

9. the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 12; the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 17 or 55; the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 22; the VL CDR1 comprises the amino acid sequence of SEQ ID NO:27; the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 31; the VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 34, 61 and 62; The antibody or antigen-binding fragment thereof according to claim 8.

10. The antibody or antigen-binding fragment thereof according to claim 9, wherein the VH comprises the amino acid sequence of SEQ ID NO: 5 and the VL comprises the amino acid sequence of SEQ ID NO:

6.

11. the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 67; the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 17 or 55; the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 23; the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 68; the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 31; the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 69; The antibody or antigen-binding fragment thereof according to claim 1.

12. the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 12; the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 17 or 55; the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 23; the VL CDR1 comprises the amino acid sequence of SEQ ID NO:27; the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 31; the VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 36, 65 and 66; The antibody or antigen-binding fragment thereof according to claim 11.

13. The antibody or antigen-binding fragment thereof according to claim 12, wherein the VH comprises the amino acid sequence of SEQ ID NO: 7 and the VL comprises the amino acid sequence of SEQ ID NO:

8.

14. the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 67; the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 17 or 55; the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 25; the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 68; the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 31; the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 69; The antibody or antigen-binding fragment thereof according to claim 1.

15. the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 12; the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 17 or 55; the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 25; the VL CDR1 comprises the amino acid sequence of SEQ ID NO:27; the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 31; the VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 34, 61 and 62; The antibody or antigen-binding fragment thereof described in claim 14.

16. The antibody or antigen-binding fragment thereof of claim 15, wherein the VH comprises the amino acid sequence of SEQ ID NO: 9 and the VL comprises the amino acid sequence of SEQ ID NO:

10.

17. 17. The antibody or fragment thereof according to any one of claims 1 to 16, which is a bivalent Fab antibody or a fragment selected from the group consisting of F(ab')2, F(ab)2, Fab', Fab, Fv and scFv.

18. A multispecific antibody comprising an antigen-binding fragment of any one of claims 1 to 16 and one or more antibodies or antigen-binding fragments having binding specificity for a target antigen that is not FAPα.

19. A chimeric antigen receptor (CAR) comprising the antigen-binding fragment of claim 1, a transmembrane domain, a costimulatory domain, and a CD3ζ intracellular domain.

20. One or more polynucleotides encoding the antibody or antigen-binding fragment thereof of any one of claims 1 to 16 or the CAR of claim 19.

21. 21. The polynucleotide of claim 20 which is one or more mRNAs.

22. 22. The polynucleotide of claim 21, wherein the mRNA is chemically modified.

23. A cell comprising the polynucleotide of claim 20.

24. A composition comprising the antibody or antigen-binding fragment thereof described in any one of claims 1 to 16, or the CAR described in claim 19, and a pharmaceutically acceptable carrier.

25. A composition for treating cancer in a patient in need thereof, comprising an antibody or antigen-binding fragment thereof described in any one of claims 1 to 16 or a CAR described in claim 19.

26. Use of the antibody or antigen-binding fragment thereof described in any one of claims 1 to 16 or the CAR described in claim 19 for the preparation of a medicament for treating cancer.

27. A composition for treating an inflammatory condition in a patient in need thereof, comprising an antibody or antigen-binding fragment thereof described in any one of claims 1 to 16.

28. 20. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 16 for the preparation of a medicament for treating an inflammatory condition.

29. 29. The use according to claim 28, wherein the inflammatory condition is associated with rheumatic rhinitis (RA).

Images