WO2019096261A1 - 靶向成纤维激活蛋白α的结合单元及其应用 - Google Patents

靶向成纤维激活蛋白α的结合单元及其应用 Download PDF

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WO2019096261A1
WO2019096261A1 PCT/CN2018/115945 CN2018115945W WO2019096261A1 WO 2019096261 A1 WO2019096261 A1 WO 2019096261A1 CN 2018115945 W CN2018115945 W CN 2018115945W WO 2019096261 A1 WO2019096261 A1 WO 2019096261A1
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seq
sequence
binding unit
antigen
antibody
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PCT/CN2018/115945
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French (fr)
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李宗海
王鹏
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科济生物医药（上海）有限公司
上海市肿瘤研究所
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Priority to EP18878002.7A priority Critical patent/EP3712179A4/en
Priority to JP2020545424A priority patent/JP2021509820A/ja
Priority to US16/765,100 priority patent/US11447570B2/en
Publication of WO2019096261A1 publication Critical patent/WO2019096261A1/zh

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    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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Definitions

  • the present invention relates to the field of biomedicine; in particular, the present invention relates to a binding unit that specifically binds to FAPa, a polynucleotide encoding such a binding unit, a vector comprising such a polynucleotide, and a host cell.
  • the invention also relates to methods for producing the antigen binding unit and methods of using the same in the treatment of a disease.
  • Fibroblast activation protein alpha is an antigen molecule specifically expressed on the surface of tumor-associated fibroblasts (CAF) (NCBI Reference Sequence: NP_001278736.1), belonging to the serine protease family, with collagenase And dipeptidyl peptidase activity plays an important role in the degradation and remodeling of tumor host interface matrix, involved in tumor growth, invasion and metastasis, and FAP ⁇ is selectively expressed in more than 90% of malignant epithelial tumors (such as breast cancer, Matrix of ovarian cancer, lung cancer, colon cancer, pancreatic cancer, cutaneous melanoma, kidney cancer, bladder cancer, etc.), embryonic tissue, healing wounds, and physiologically reconstructed organs, but not normally expressed in normal adult tissues. These characteristics make FAP ⁇ an antigenic target for imaging, diagnosis, and treatment of a variety of tumors.
  • CAF tumor-associated fibroblasts
  • the present invention aims to find binding units that specifically bind to FAPa, including but not limited to antibodies and development of immune effector cells that target FAPa.
  • the invention provides an antigen binding unit comprising a light chain CDR region comprising a HCDR1, an HCDR2, and an HCDR3, and a heavy chain CDR region; said light chain CDR
  • the area includes LCDR1, LCDR2, and LCDR3;
  • sequences of HCDR1, HCDR2 and HCDR3 are independently selected from the group consisting of SEQ ID NOs: 1-3, 7 and 8, wherein the sequences of LCDR1, LCDR2 and LCDR3 are independently selected from the group consisting of SEQ ID NO: 4-6, 9 and 10.
  • said HCDR1 has the sequence set forth in SEQ ID NO: 1 or 7, said HCDR2 having the sequence set forth in SEQ ID NO: 2 or 8, said HCDR3 having SEQ ID NO: The sequence shown in 3.
  • sequences of HCDR1, HCDR2 and HCDR3 are selected from the group consisting of:
  • SEQ ID NO: 1 SEQ ID NO: 2, SEQ ID NO: 3;
  • the LCDR1 has the sequence shown in SEQ ID NO: 4 or 9
  • the LCDR2 has the sequence shown in SEQ ID NO: 5 or 10
  • the LCDR3 has the SEQ ID NO: The sequence shown in 6.
  • the sequence of the LCDR1, LCDR2, and LCDR3 is selected from any one of the following groups:
  • SEQ ID NO: 4 SEQ ID NO: 5;
  • said HCDR1 has the sequence set forth in SEQ ID NO: 1 or 7, said HCDR2 having the sequence set forth in SEQ ID NO: 2 or 8, said HCDR3 having SEQ ID NO: The sequence shown in 3, wherein the LCDR1 has the sequence shown in SEQ ID NO: 4 or 9, the LCDR2 has the sequence shown in SEQ ID NO: 5 or 10, and the LCDR3 has SEQ ID NO: 6. The sequence shown.
  • the HCDR1 comprises the sequence set forth in SEQ ID NO: 1
  • the HCDR2 comprises the sequence set forth in SEQ ID NO: 2
  • the HCDR3 comprises the sequence set forth in SEQ ID NO: a sequence
  • said LCDR1 comprises the sequence of SEQ ID NO: 4
  • said LCDR2 comprises the sequence of SEQ ID NO: 5
  • said LCDR3 comprises the sequence of SEQ ID NO: 6;
  • the HCDR1 comprises the sequence of SEQ ID NO: 7
  • the HCDR2 comprises the sequence of SEQ ID NO: 8
  • the HCDR3 comprises the sequence of SEQ ID NO: 3
  • the LCDR1 contains a sequence represented by SEQ ID NO: 4, wherein the LCDR2 comprises the sequence of SEQ ID NO: 5, and the LCDR3 comprises the sequence of SEQ ID NO: 6;
  • the HCDR1 comprises the sequence of SEQ ID NO: 1
  • the HCDR2 comprises the sequence of SEQ ID NO: 2
  • the HCDR3 comprises the sequence of SEQ ID NO: 3
  • the LCDR1 contains The sequence shown in SEQ ID NO: 9
  • the LCDR2 contains the sequence shown in SEQ ID NO: 10
  • the LCDR3 contains the sequence shown in SEQ ID NO: 6.
  • the antigen binding unit has the heavy chain variable region set forth in SEQ ID NO: 11 or 15, and the light chain variable region set forth in SEQ ID NO: 13 or 17.
  • the antigen binding unit is an antibody, preferably a monoclonal antibody or an active fragment thereof.
  • the invention provides a mutant of the antigen binding unit of the first aspect, the mutant which competes with the antigen binding unit of the first aspect for binding to FAPa.
  • the mutant and the antigen binding unit of the first aspect are 90%, 91%, 92%, 93%, 94%, 95%, 96% of the light chain variable region sequence. , 97%, 98%, or 99% identity; or, the antigen and the antigen binding unit of any of claims 1-8 are 90%, 91%, 92 in the heavy chain variable region sequence. %, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity;
  • the mutant and the antigen binding unit of the first aspect are 70% or more in the sequence of the light chain CDR region, such as the light chain CDR1 or CDR2; preferably, 75% or more; more preferably, 80% The above identity; or the antigen and the antigen binding unit of the first aspect are 60% or more; preferably 70% or more; more preferably in the sequence of the heavy chain CDR region, such as the heavy chain CDR1 or CDR2; Ground, more than 80% identity.
  • the mutant competes with the antigen binding unit of the first aspect for binding to the same binding epitope on FAPa.
  • the invention provides an antigen binding unit comprising a light chain CDR region comprising a HCDR1, an HCDR2 and an HCDR3, and a heavy chain CDR region; said light chain CDR region Included are LCDR1, LCDR2 and LCDR3, wherein said HCDR1, HCDR2 and HCDR3 are independently selected from at least 80%, preferably at least 85%, more preferably at least 90% of the sequence of any one of SEQ ID NOs: 1-3, 7, 8.
  • the sequence is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% similar
  • the LCDR1, LCDR2 and LCDR3 are independently selected from the group consisting of SEQ ID NO : any of 4-6, 9, 10 is at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98% or 99% similarity of the sequence.
  • said HCDR1 has the sequence set forth in SEQ ID NO: 1 or 7, said HCDR2 having the sequence set forth in SEQ ID NO: 2 or 8, said HCDR3 having SEQ ID NO: The sequence shown in 3.
  • sequences of HCDR1, HCDR2 and HCDR3 are selected from the group consisting of:
  • SEQ ID NO: 1 SEQ ID NO: 2, SEQ ID NO: 3;
  • the LCDR1 has the sequence shown in SEQ ID NO: 4 or 9
  • the LCDR2 has the sequence shown in SEQ ID NO: 5 or 10
  • the LCDR3 has the SEQ ID NO: The sequence shown in 6.
  • the sequence of the LCDR1, LCDR2, and LCDR3 is selected from any one of the following groups:
  • SEQ ID NO: 4 SEQ ID NO: 5;
  • said HCDR1 has the sequence set forth in SEQ ID NO: 1 or 7, said HCDR2 having the sequence set forth in SEQ ID NO: 2 or 8, said HCDR3 having SEQ ID NO: The sequence shown in 3, wherein the LCDR1 has the sequence shown in SEQ ID NO: 4 or 9, the LCDR2 has the sequence shown in SEQ ID NO: 5 or 10, and the LCDR3 has SEQ ID NO: 6. The sequence shown.
  • the HCDR1 comprises the sequence set forth in SEQ ID NO: 1
  • the HCDR2 comprises the sequence set forth in SEQ ID NO: 2
  • the HCDR3 comprises the sequence set forth in SEQ ID NO: a sequence
  • said LCDR1 comprises the sequence of SEQ ID NO: 4
  • said LCDR2 comprises the sequence of SEQ ID NO: 5
  • said LCDR3 comprises the sequence of SEQ ID NO: 6;
  • the HCDR1 comprises the sequence of SEQ ID NO: 7
  • the HCDR2 comprises the sequence of SEQ ID NO: 8
  • the HCDR3 comprises the sequence of SEQ ID NO: 3
  • the LCDR1 contains a sequence represented by SEQ ID NO: 4, wherein the LCDR2 comprises the sequence of SEQ ID NO: 5, and the LCDR3 comprises the sequence of SEQ ID NO: 6;
  • the HCDR1 comprises the sequence of SEQ ID NO: 1
  • the HCDR2 comprises the sequence of SEQ ID NO: 2
  • the HCDR3 comprises the sequence of SEQ ID NO: 3
  • the LCDR1 contains The sequence shown in SEQ ID NO: 9
  • the LCDR2 contains the sequence shown in SEQ ID NO: 10
  • the LCDR3 contains the sequence shown in SEQ ID NO: 6.
  • the present invention provides an antigen binding unit having at least 80%, preferably at least 85%, more preferably the sequence of the heavy chain variable region of SEQ ID NO: 11 or 15 a sequence of at least 90%, more preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% similarity, and as shown in SEQ ID NO: 13 or 17
  • Light chain variable region having at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99 Sequence of % similarity.
  • the present invention provides the antigen-binding unit or the antigen-binding unit mutant according to the first to fourth aspect, wherein the antigen-binding unit is a monoclonal antibody, a fully human antibody, a humanized antibody, or a chimeric antibody. .
  • the antigen binding unit is a scFv, Fv, Fab, (Fab) 2 or a single domain antibody.
  • the invention provides a nucleic acid encoding the antigen binding unit of the first to fifth aspects.
  • the present invention provides an expression vector comprising the nucleic acid of the sixth aspect.
  • the present invention provides a host cell comprising the expression vector of the seventh aspect or the nucleic acid according to the sixth aspect integrated in the genome.
  • the present invention provides a bivalent protein, wherein the scFv sequence of the fifth aspect is fused to one or more heavy chain constant regions to form an antibody having a human immunoglobulin Fc region;
  • the human immunoglobulin Fc region is linked to a conjugate; preferably, the conjugate is preferably a fluorescent dye, a cytotoxin, a radioisotope.
  • the present invention provides a multifunctional immunoconjugate comprising:
  • a molecule that targets a tumor surface marker including an antibody or ligand that binds to an immunocyte surface marker
  • the immune cell surface marker comprises: CD3, CD16, CD28; more preferably, The antibody that binds to an immunocyte surface marker is an anti-CD3 antibody; optimally, the molecule that targets a surface marker of an immune cell is an antibody that binds to a T cell surface marker;
  • the cytokine comprises: IL-12, IL-15, type I interferon, TNF-alpha;
  • the present invention provides a chimeric antigen receptor, wherein the extracellular domain of the chimeric antigen receptor comprises the antigen binding unit of the first to fifth aspects, and the antigen binding unit is preferably a single chain antibody or Single domain antibody.
  • the chimeric antigen receptor further comprises a transmembrane domain and an intracellular signal domain
  • the transmembrane domain preferably, is selected from the group consisting of ⁇ , ⁇ , zeta chains of TCR, CD3 ⁇ , CD3 ⁇ , CD4, CD5, CD8 ⁇ , CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, a transmembrane region of CD80, CD86, CD134, CD137, CD152, CD154, and PD1; more preferably, a transmembrane domain selected from the group consisting of CD8 ⁇ , CD4, CD45, PD1, CD154, and CD28;
  • the intracellular signal domain comprises one or more costimulatory signal domains and/or a primary signal domain; wherein the costimulatory signal domain, preferably selected from the group consisting of CARD11, CD2, CD7, CD27, CD28, CD30, Intracellular signal regions of CD40, CD54, CD83, OX40, CD137, CD134, CD150, CD152, CD223, CD270, PD-L2, PD-L1, CD278, DAP10, LAT, NKD2C SLP76, TRIM, Fc ⁇ RI ⁇ , MyD88, and 41BBL
  • the primary signal domain is selected from the group consisting of TCR ⁇ , FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD79a, CD79b, CD278 (also referred to as “ICOS”) and CD66d, and CD3 ⁇ ; more preferably, Selected from CD137, CD134, CD28 and OX40; and/or the primary signal domain is selected from the group
  • the transmembrane domain is selected from the group consisting of CD8 ⁇ or CD28
  • the costimulatory signal domain is selected from the intracellular signal domain of CD137 or CD28
  • the primary signal domain is selected from the group consisting of CD3 ⁇ .
  • the chimeric antigen receptor comprises the following sequentially linked antibodies, a transmembrane region and an intracellular signal region:
  • the antigen binding unit of the first to fifth aspects CD8, CD137 and CD3 ⁇ ;
  • the antigen binding unit of the first to fifth aspects the transmembrane region of the CD28 molecule, the intracellular signal region of the CD28 molecule, and CD3 ⁇ ; or
  • the antigen-binding unit of the first to fifth aspects the transmembrane region of the CD28 molecule, the intracellular signal region of the CD28 molecule, CD137 and CD3 ⁇ .
  • the invention provides a nucleotide sequence encoding the chimeric antigen receptor of the eleventh aspect.
  • the present invention provides an expression vector comprising the nucleotide sequence of the twelfth aspect.
  • the present invention provides a virus comprising the expression vector of the thirteenth aspect.
  • the present invention provides a genetically modified immune cell, which has the nucleotide sequence of the twelfth aspect, or the expression vector of the thirteenth aspect, or the fourteenth aspect Said virus; or it expresses the chimeric antigen receptor described in the eleventh aspect.
  • the immune cells include, but are not limited to, the following cells and combinations thereof: T lymphocytes, NK cells or NKT cells.
  • the genetically modified immune cell further expresses a sequence other than a chimeric antigen receptor, including a cytokine, another chimeric antigen receptor, and a chemokine receptor.
  • a siRNA that reduces PD-1 expression or a protein, TCR, or a safety switch that blocks PD-L1;
  • the cytokine comprises IL-12, IL-15, IL-21, or type I interferon;
  • the chemokine receptor comprises CCR2, CCR5, CXCR2, or CXCR4;
  • the safety switch comprises iCaspase-9, Truncated EGFR or RQR8.
  • the present invention provides the antigen-binding unit according to the first to fifth aspects, or the bisvalent protein according to the ninth aspect, or the immunoconjugate according to the tenth aspect, or the eleventh aspect
  • the chimeric antigen receptor, or the nucleotide sequence of the twelfth aspect, or the expression vector of the thirteenth aspect, or the virus of the fourteenth aspect, or the fifteenth aspect Use of immune cells for the preparation of a medicament or agent for the treatment, prevention or diagnosis of a disease which is highly expressed in FAPa.
  • the high expression of FAPa associated disease is a tumor.
  • the tumor is breast cancer, ovarian cancer, lung cancer, colon cancer, pancreatic cancer, cutaneous melanoma, kidney cancer, bladder cancer.
  • the present invention provides a pharmaceutical composition comprising the antigen-binding unit of the first to fifth aspects or a nucleic acid encoding the antigen-binding unit; or
  • a chimeric antigen receptor according to the eleventh aspect or a nucleic acid encoding the chimeric antigen receptor;
  • the genetically modified immune cell of the fifteenth aspect is provided.
  • Figure 1 shows the results of SDS PAGE analysis of nickel column purified recombinant human FAP ⁇ _His (huFAP), mouse FAP_His (mFAP);
  • Figure 2 shows the ELISA assay for binding of phage clone expression supernatant to recombinant huFAPa, mFAP;
  • Figure 3 shows FACs detecting the binding of partially purified clones to U-87MG and A549 cells
  • Figure 4 shows the affinity of Biacore assay 1A7, 8E3 (scFv) binding to huFAP, mFAP;
  • Figure 5 shows the sequence alignment (scFv) of antibody 1A7, 8E3 and parent antibody 10A4;
  • Figure 6 is a purified antibody 1A7, 8E3 (scFv_Fc) by SDS PAGE analysis;
  • Figure 7 shows the results of Western blot analysis of 3T3-mFAP, HT1080-huFAP ⁇ stably transfected cell lines
  • Figure 8 shows the results of FACs detecting 3T3-mFAP(a), HT1080-huFAP ⁇ (b) stable cell lines;
  • Figure 9 shows FACs assay binding of antibodies 10A4, 1A7, 8E3 (scFv_Fc) to U-87MG, 3T3-mFAP, HT1080-huFAP ⁇ cells;
  • Figure 10 is a diagram showing the expression of mFAP ⁇ and huFAP ⁇ in 3T3-mFAP(a), HT1080-huFAP(b) stably transfected cell lines using Anti-Flag antibody FACs;
  • Figure 11 is a FACs assay for the expression of hu8E3-28Z CAR in T cells
  • Figure 12 is a graph showing the killing of 3T3-mFAP (a) and H1080-huFAP ⁇ (b) cells by T cells expressing hu8E3-28Z CAR by cytotoxicity.
  • the inventors have extensively and intensively studied and unexpectedly discovered antibodies that specifically bind to FAP, and on this basis, developed immune effector cells that specifically target FAP.
  • the antibody of the present invention can effectively bind to tumor cells expressing FAP, and the immune effector cells of the present invention exhibit remarkable killing ability against tumor cells expressing FAP, thereby being effectively and safely applied to the treatment of malignant tumors such as multiple myeloma.
  • the present invention has been completed on this basis.
  • FAP ⁇ refers to fibroblast activation protein alpha.
  • Human fibroblast activation protein alpha is a transmembrane protein consisting of 760 amino acid residues (NCBI Reference Sequence: NP_001278736.1). If not specified, FAP ⁇ herein refers to human fibroblast activation protein alpha.
  • the murine fibroblast activation protein consists of 761 amino acid residues (transmembrane protein consisting of 760 amino acid residues, NCBI Reference Sequence: NP_032012.1).
  • binding unit As used herein have the same meaning and are used interchangeably herein to refer to any form of immunologically active portion of an immunoglobulin molecule and an immunoglobulin molecule. That is, a molecule comprising an antigen binding site that specifically binds to an antigen or immunoreacts with an antigen. Structurally, the simplest naturally occurring antibody (eg, IgG) has four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Immunoglobulins represent a large family of molecules including several molecular types, such as IgD, IgG, IgA, IgM, and IgE.
  • Antigen binding units include, but are not limited to, Fv, scFv, dFv, dAb, diabodies, triabodies, tetra-chain antibodies, domain Ab, Fab fragments, Fab', (Fab') 2 , bispecific antibodies, and multispecific Sexual antibodies.
  • binding refers to the same position of two or more substances to a binding partner, for example, the same binding epitope of an antigen; in other words, different substances bound at different positions on the same binding object do not exist.
  • antigen binding unit also includes immunoglobulin molecules of multi-species origin, including invertebrates and vertebrates.
  • human as applied to an antigen binding unit refers to an immunoglobulin molecule expressed by a human gene or a fragment thereof.
  • humanized applied to a non-human (eg, rodent or primate) antibody is a hybrid immunoglobulin, immunoglobulin chain or fragment thereof comprising a minimal sequence derived from a non-human immunoglobulin.
  • the humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the complementarity determining regions (CDRs) of the recipient are derived from a non-specificity, affinity and capacity.
  • CDRs complementarity determining regions
  • Residue substitution of the CDR (donor antibody) of a human species such as mouse, rat, rabbit or primate.
  • the Fv framework region (FR) residues of the non-human immunoglobulin are replaced by corresponding human residues.
  • a humanized antibody can comprise residues that are neither found in the recipient antibody nor in the CDR or framework sequences of the input.
  • a humanized antibody comprises substantially all of at least one and typically two variable domains, wherein all or substantially all of the CDR regions correspond to CDR regions of a non-human immunoglobulin and all or substantially all of the FR regions are The FR region of a non-human immunoglobulin sequence.
  • antibody refers to an antigen binding protein of the immune system.
  • antibody as referred to herein includes intact full length antibodies having an antigen binding region and any fragments thereof, or a single strand thereof, such as a single chain variable fragment, retained by an "antigen-binding portion” or “antigen-binding region” thereof ( scFv).
  • a native antibody refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains or antigen-binding fragments thereof interconnected by a disulfide bond.
  • antibody also includes all recombinant forms of antibodies, particularly the antibodies described herein, such as antibodies expressed in prokaryotic cells, unglycosylated antibodies, and antibody fragments that bind to antigens and derivatives hereinafter.
  • Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH ) and a heavy chain constant region.
  • Each light chain is comprised of a light chain variable region (abbreviated herein as V L) and a light chain constant region.
  • V H and V L can be further subdivided termed complementarity determining region (CDR) of the hypervariable regions, they are scattered in more conserved, termed framework regions (FR) of.
  • CDR complementarity determining region
  • variable regions of the heavy and light chains contain a binding domain that interacts with the antigen.
  • the constant region of the antibody can mediate binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and the first component (C1q) of the classical complement system.
  • Fab fragment or "Fab region” includes V H, polypeptide C H1, V L and C L immunoglobulin domains.
  • a Fab can refer to this region of isolation, or to that region in the context of a full length antibody or antibody fragment.
  • Fc or "Fc region” as used herein includes a polypeptide comprising an antibody constant region other than the first constant region immunoglobulin domain.
  • Fc refers to the last two constant region immunoglobulin domains of IgA, IgD and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinges at the N-terminus of these domains.
  • IgA and IgM Fc can include a J chain.
  • Fc includes the immunoglobulin domains C ⁇ 2 and C ⁇ 3 and the hinge between C ⁇ 1 and C ⁇ 2.
  • the human IgG heavy chain Fc region is generally defined as comprising residues C226 or P230 at its carboxy terminus, where numbering is according to the EU index of Kabat.
  • Fc is defined herein to include residue P232 to its carboxy terminus, where numbering is based on the EU index in Kabat.
  • Fc may refer to this region of isolation, or to that region of an Fc polypeptide, such as an antibody, environment.
  • parent antibody or “parent immunoglobulin” as used herein includes unmodified antibodies which are then modified to produce variants.
  • the parent antibody can be a naturally occurring antibody, or a variant or modified version of a naturally occurring antibody.
  • a parent antibody can refer to the antibody itself, a composition comprising the parent antibody, or a nucleic acid sequence encoding the same.
  • parent antibody or parent immunoglobulin as used herein includes murine or chimeric antibodies that are subsequently modified to produce a humanized antibody.
  • variable antibody or “antibody variant” or “variant” as used herein includes an antibody sequence that differs from the parent antibody sequence due to at least one amino acid modification compared to the parent.
  • An antibody variant may refer to the antibody itself, and may also refer to a composition comprising the parent antibody.
  • the light chain variable region sequence of the variant antibody described herein is 90%, 91%, 92%, 93%, 94%, 95% of the light chain variable region sequence of the parent antibody, 96%, 97%, 98%, or 99% identity;
  • the heavy chain variable region sequences of the variant antibodies described herein are 90%, 91%, 92%, 93 of the heavy chain variable region sequences of the parent antibody.
  • the light chain CDR regions of the variant antibodies described herein are 70% or more identical to the light chain CDR regions of the parent antibody, such as the light chain CDR1 or CDR2 sequences.
  • the heavy chain CDR regions of the variant antibodies described herein are more than 60% identical; preferably, more than 70% identical; more preferably, more than 80% identical.
  • amino acid modification includes amino acid substitutions, additions and/or deletions
  • amino acid substitution means the replacement of an amino acid at a particular position in the parent polypeptide sequence with another amino acid.
  • substituting R94K means that the arginine at position 94 is replaced by lysine
  • amino acid insertion means the addition of an amino acid at a particular position in the parent polypeptide sequence.
  • amino acid deletion or “deletion” means removal of an amino acid at a particular position in the parent polypeptide sequence.
  • conservative modification or “conservative sequence modification” as used herein means an amino acid modification that does not significantly affect or alter the binding characteristics of an antibody comprising the amino acid sequence. Such conservative modifications include amino acid substitutions, insertions, and deletions. Modifications can be introduced into the antibodies of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are substitutions in which amino acid residues are replaced with amino acid residues having similar side chains. A family of amino acid residues having similar side chains has been defined in the art.
  • amino acids containing basic side chains eg, lysine, arginine, histidine
  • acidic side chains eg, aspartic acid, glutamic acid
  • uncharged polar side chains eg, glycine, asparagine, serine, threonine, tyrosine, cysteine, tryptophan
  • non-polar side chains eg, alanine, valine, leucine, isoluminescence
  • Acid valine, phenylalanine, methionine
  • beta branch side chains eg, threonine, valine, isoleucine
  • aromatic side chains eg, tyrosine, Phenylalanine, tryptophan, histidine
  • Antibody fragments include but are not limited to: (i) Fab fragment consisting of V L, V H, C L and C H1 domains including Fab 'and Fab'-SH, (ii) V H and C H1 domains of a Fd fragment, (iii) Fv fragment consisting of the V L and V H domains of a single antibody composition; (iv) dAb fragment consisting of a single variable domain (Ward et al, 1989, Nature 341: 544-546) ; (v F(ab') 2 fragment comprising a bivalent fragment of two linked Fab fragments; (vi) single-chain Fv molecule antigen binding site (Bird et al, 1988, Science 242: 423-426; Huston et al, 1988, Proc.
  • Antibodies are classified according to constant region gene assays, also referred to as isotypes.
  • the human constant light chain is divided into ⁇ (C ⁇ ) and ⁇ (C ⁇ ) light chains.
  • Heavy chains are classified as mu, delta, gamma, alpha or epsilon and define the isotypes of antibodies IgM, IgD, IgG, IgA and IgE, respectively.
  • IgG classes are most commonly used for therapeutic purposes. In humans, this category includes subclasses IgG1, IgG2, IgG3, and IgG4. In mice, this category includes subclasses IgG1, IgG2a, IgG2b, and IgG3.
  • IgM has subclasses including, but not limited to, IgM1 and IgM2.
  • IgA has several subclasses including, but not limited to, IgA1 and IgA2.
  • isotype means any class or subclass of immunoglobulin defined by the chemical and antigenic characteristics of the constant region.
  • the known human immunoglobulin isotypes are IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM1, IgM2, IgD and IgE.
  • antigen binding proteins having an antigen-binding region based on scFv, including antibodies, are described. Where scFv was selected from a human scFv phage display library using recombinant FAPa.
  • the invention encompasses an antibody having a scFv sequence fused to one or more heavy chain constant regions to form an antibody having a human immunoglobulin Fc region to produce a bivalent protein, thereby increasing antibody Overall affinity and stability.
  • the Fc portion allows for direct conjugation of other molecules (including but not limited to fluorescent dyes, cytotoxins, radioisotopes, etc.) to, for example, antibodies used in antigen quantification studies in order to immobilize antibodies for affinity measurement, for targeted delivery therapy. Drugs, use of immune effector cells to test Fc-mediated cytotoxicity and many other applications.
  • the molecules of the invention are based on the use of phage display to identify and select single-chain variable fragments (scFv) whose amino acid sequence confers on the molecule specificity for FAPa and forms the basis of all antigen binding proteins of the invention.
  • the scFv can be used to design a range of different "antibody” molecules including, for example, full length antibodies, fragments thereof such as Fab and F(ab') 2 , fusion proteins (including scFv_Fc), multivalent antibodies, ie, having More than one specific antibody of the same antigen or different antigens, for example, bispecific T cell binding antibody (BiTE), triabodies, etc.
  • BiTE bispecific T cell binding antibody
  • the heavy and light chains of the antibodies of the invention may be full length (eg, the antibody may comprise at least one and preferably two intact heavy chains, and at least one and preferably The two intact light chains) may alternatively comprise an antigen binding moiety (Fab, F(ab') 2 , Fv or scFv).
  • the antibody heavy chain constant region is selected, for example, from IgGl, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE. The choice of antibody type will depend on the immune effector function that the designed antibody is intended to elicit. Suitable amino acid sequences for the constant regions of various immunoglobulin isotypes and methods for producing a wide variety of antibodies are known to those skilled in the art in the construction of recombinant immunoglobulins.
  • the invention provides an antigen binding unit that binds FAPa, comprising a heavy chain variable region sequence selected from the group consisting of SEQ ID NOs: 11, 15.
  • the invention provides an antigen binding unit that binds to FAP ⁇ , comprising a light chain variable region sequence selected from the group consisting of SEQ ID NOs: 13 and 17.
  • the heavy and light chain variable region sequences can bind to FAPa
  • the heavy and light chain variable region sequences can be "mixed and matched" to produce the anti-FAP[alpha] binding molecules of the invention.
  • the invention provides a variant of an antibody or fragment thereof that binds FAPa.
  • the invention thus provides an antibody or fragment thereof having a heavy chain and/or light chain variable region that is at least 80% identical to the variable region sequence of the heavy or light chain of an antibody of the invention.
  • the amino acid sequence identity of the heavy and/or light chain variable regions is at least 85%, more preferably at least 90%, most preferably at least 95%, especially 96%, more particularly 97%, even more particularly 98%.
  • the most special 99% including, for example, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100%.
  • Standard assays for assessing the binding capacity of antibodies, such as antibodies against FAP are known in the art and include, for example, ELISA, Biacore, Western blot, and flow cytometry analysis. Suitable assays are described in detail in the examples.
  • the invention also provides nucleic acids, vectors, and host cells comprising the nucleic acid or vector, which encode an antibody that binds to FAP ⁇ and a fragment thereof.
  • the nucleic acid can be located in intact cells, in cell lysates, or in partially purified or substantially purified form.
  • the nucleic acids of the invention can be obtained using standard molecular biology techniques, for example, by standard PCR amplification or cDNA cloning techniques, obtaining light and heavy chains encoding antibodies or cDNA encoding VH and VL segments.
  • standard molecular biology techniques for example, by standard PCR amplification or cDNA cloning techniques, obtaining light and heavy chains encoding antibodies or cDNA encoding VH and VL segments.
  • antibodies obtained from immunoglobulin gene libraries eg, using phage display technology
  • one or more nucleic acids encoding the antibodies can be recovered from the library. Methods for introducing foreign nucleic acids into host cells are generally known in the art and can vary with the host cell used.
  • a nucleic acid encoding an antibody of the invention can be integrated into an expression vector.
  • expression vectors are available for protein expression.
  • Expression vectors can include self-replicating extra-chromosomal vectors, or vectors integrated into the host genome.
  • Expression vectors for use in the present invention include, but are not limited to, those which enable expression of proteins in mammalian cells, bacteria, insect cells, yeast, and in vitro systems. As is known in the art, a variety of expression vectors are commercially available or otherwise available. It can be used in the present invention to express antibodies.
  • the invention provides an antibody that specifically binds to FAPa
  • the invention provides immune effector cells that specifically target FAP ⁇ ;
  • the antibody of the present invention is capable of efficiently binding to tumor cells expressing FAPa, and the immune effector cells of the present invention exhibit significant killing ability against tumor cells expressing FAP.
  • the extracellular domain Leu26-Asp760 gene of human FAP ⁇ was synthesized in vitro, and the His tag was inserted into the C-terminus of the gene, and the GS was ligated in the middle to form the fusion expression protein human FAP ⁇ _His (SEQ ID NO: 19).
  • the corresponding gene sequence was as follows. SEQ ID NO:20.
  • the in vitro synthesized mouse FAP extracellular domain gene (Leu26-Asp761) was inserted into the His-tagged C-terminus and ligated with "GS" in the middle to form the fusion expression protein mouse FAP ⁇ _His (SEQ ID NO: 21).
  • the corresponding gene sequence is SEQ ID. NO: 22 is shown.
  • Human FAP ⁇ _His and mouse FAP ⁇ _His were transiently transfected with 293F cells, respectively.
  • the method of transfection was carried out by referring to the instruction manual of 293F cells. After transfection, affinity purification was performed using a nickel column.
  • concentration was carried out using a millipore ultrafiltration tube having a cross-sectional flow rate of 10 KD, and concentrated to a volume of 1 ml or less, and desalted using a PD-Midi desalting column to collect human and mouse FAPa recombinant protein.
  • Example 2 Screening for scF-specific scFv using a fully human phage display library
  • the phage display library used in the present invention is a fully human native scFv phage library constructed by the applicant, having a library capacity of 1E+11. Using a screening method known to those skilled in the art, 10 ug/ml of antigen human FAP ⁇ was coated in an immunotube, and the phage library was added to an immunotube coated with human FAP ⁇ for 1.5 hours to screen and enrich for specific binding to FAPa. scFv phage clone.
  • FACs detected U-87MG cells endogenously expressing FAP ⁇ (purchased from the cell bank of the Chinese Academy of Sciences), and A549 cells (purchased from the Chinese Academy of Sciences cell bank) that did not express FAPa were used as negative control cells.
  • the results were detected using a Guava easyCyteTM HT System instrument, and the results are shown in Figure 3.
  • Antibody 10A4 specifically binds to U-87MG cells.
  • the heavy chain variable region of 10A4 is the sequence shown in SEQ ID NO: 11, and the sequences of HCDR1, HCDR2, and HCDR3 of the heavy chain are shown in SEQ ID NOs: 1, 2, and 3, respectively.
  • the light chain variable region of 10A4 is the sequence shown in SEQ ID NO: 13, and the sequences of the light chain LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NOs: 4, 5, and 6, respectively.
  • the template plasmid was first constructed based on the scFv of the antibody 10A4 (amino acid sequence SEQ ID NO: 23, nucleotide sequence SEQ ID NO: 24).
  • fragment 1 was PCR amplified using primers LMF (SEQ ID NO: 41) and F10L1R (SEQ ID NO: 45); primers F10L2F (SEQ ID NO: 46) and FdR (using primers) SEQ ID NO: 44) PCR amplification of fragment 2; ligation of fragment 1 and fragment 2 by bridge PCR to obtain the full length of the scFv containing the randomized sequence, followed by digestion of the full-length fragment with NcoI and NotI, and ligation of the same enzyme by T4 ligase Cut the template in the plasmid.
  • fragment 3 was PCR amplified using primers LMF (SEQ ID NO: 41) and F10H1R (SEQ ID NO: 42); primers F10H2F (SEQ ID NO: 43) and FdR were used. (SEQ ID NO: 44), PCR amplified fragment 4; then ligated to fragment 3 and fragment 4 by bridging PCR to obtain the full length of the scFv containing the randomized sequence, followed by digestion of the full-length fragment with NcoI and NotI, and ligated by T4 ligase Into the same digested template plasmid. And electroporation into TG1 competent cells, the storage capacity is 6.6E+8.
  • the screening of the affinity mature library was substantially identical to the description in Example 2.
  • the antigen was coated with 5 ug/ml or 1 ug/ml of antigen human FAP ⁇ in the immunotube.
  • the phage library was added to the immunotube coated with human FAP ⁇ for 1.5 hours. After screening and enrichment, the scFv phage clone which specifically binds to human FAP ⁇ was obtained. .
  • primers were designed 10A4,1A7,8E3 fragments, the introduction of a flexible linker of 15 amino acids (GGGGSGGGGSGGGGS) connected composition consisting of scFv (10A4, SEQ ID NO: 23; 1A7, SEQ ID NO: 25; 8E3 , SEQ ID NO: 27); the V H and the restriction sites introduced upstream protective base Nhe I, the introduction of restriction sites BamHI and protection bases downstream of the L V.
  • the PCR product was analyzed by 1% agarose gel electrophoresis and purified and recovered.
  • the full-length sequence of huFAPa was synthesized in vitro, and the Flag tag (SEQ ID NO: 29) was inserted at the C-terminus, and double-digested with MluI/SalI, and inserted into the same double-digested lentiviral packaging plasmid pWPT.
  • the full-length sequence of mFAPa was synthesized in vitro, and the Flag tag (SEQ ID NO: 30) was inserted at the C-terminus, and double-digested with MluI/SalI, and inserted into the same double-digested lentiviral packaging plasmid pWPT.
  • the plasmid mixture was added dropwise to the PEI incubation solution, and allowed to stand at room temperature for 20 minutes.
  • the configured plasmid/PEI mixture was then added dropwise to the cells.
  • the medium was changed to fresh complete medium.
  • the cell culture supernatant was collected, centrifuged at 4 °, 3000 rpm for 15 minutes, and the supernatant was filtered through a 0.45 um filter to obtain an mFAP virus solution.
  • the preparation method of huFAP virus liquid is the same as the preparation of mFAP virus liquid.
  • HT1080 cells were infected with huFAP ⁇ virus, and 3T3 cells were infected with mFAP virus solution.
  • 3T3-huFAP mixed clonal cells or HT1080-huFAP ⁇ mixed cloned cells were lysed by protein lysate. After centrifugation, the supernatant was taken for BCA concentration, and 3T3 was used as a negative control.
  • the mixed cloned cells of HT1080-huFAP ⁇ and 3T3-mFAP were separately digested and counted, and subjected to monoclonal plating by limiting dilution method to obtain stable cell lines of HT1080-huFAP ⁇ and 3T3-mFAP.
  • Flow detection was performed using 4 ⁇ 105 cells, respectively, and detected using a Guava easyCyteTM HT System instrument. As a result, as shown in Fig. 8, the expression of FAP was detected in both stable cell lines.
  • the anti-Flag antibody was used to detect 3T3-mFAP, HT1080-huFAP ⁇ cell line, and 1 ⁇ 10 6 cells were used for flow detection. The results are shown in Figure 10. By detecting the Flag tag carried on the FAP, the two strains are stabilized. The expression of FAP was detected in the transgenic cell line.
  • Example 7 FACs assay binding of scFv_Fc of antibodies 10A4, 1A7, 8E3 to U-87MG, 3T3-mFAP, HT1080-huFAP ⁇ cells
  • 3T3 cells and HT1080 cells were used as negative control cells.
  • the specific steps are: harvesting the cells, washing the cells once with the growth medium, resuspending in PBS, and adjusting the cell concentration to 4E+5 cells/ml.
  • the 200 nM antibody was incubated with the cells for 30 minutes on ice. Thereafter, it was incubated with a FITC-labeled anti-human IgG secondary antibody. After two washes, it was tested using a Guava easyCyteTM HT System instrument.
  • antibody 10A4 was able to bind to the HT1080 cell line stably transfected with huFAP ⁇ , stably transfected with the mTAP 3T3 cell line, and endogenously expressed huFAP ⁇ -expressing U-87MG cells, but not with HT1080 cells that did not express huFAP ⁇ .
  • Antibody 8E3 did not bind to the negative cell lines 3T3 and HT1080, but bound to the cell lines positively expressing FAP, HT1080-huFAP ⁇ , 3T3-mFAP, and U-87MG.
  • Antibody 1A7 and the negative cell lines 3T3 and HT1080 did not bind, and both HT1080-huFAP ⁇ and 3T3-mFAP bound, but did not bind to U-87MG cells.
  • Example 8 Preparation of chimeric antigen receptor (CAR) modified T cells targeting FAP ⁇
  • PRRLSIN-cPPT.EF-1 ⁇ a lentiviral plasmid expressing the second-generation chimeric antigen receptors of antibodies 10A4, 1A7, and 8E3 was constructed, including PRRLSIN-cPPT.EF-1 ⁇ -10A4-28Z and PRRLSIN-cPPT. EF-1 ⁇ -1A7-28Z and PRRLSIN-cPPT.EF-1 ⁇ -8E3-28Z.
  • the 10A4-28Z sequence consists of CD8 ⁇ signal peptide (SEQ ID NO: 32), 10A4 scFv (SEQ ID NO: 24), CD8hinge (SEQ ID NO: 34), CD28 transmembrane region (SEQ ID NO: 36), and intracellular signaling
  • the 1A7-28Z sequence consists of the CD8 ⁇ signal peptide (SEQ ID NO: 32), 1A7scFv (SEQ ID NO: 26), CD8hinge (SEQ ID NO: 34), CD28 transmembrane region (SEQ ID NO: 36) and intracellular signaling domain (SEQ ID NO: 38), and the intracellular segment CD3 (CD ID NO: 40) of CD3;
  • the 8E3-28BB sequence consists of a CD8 ⁇ signal peptide (SEQ ID NO: 32), 8E3-scFv (SEQ ID NO:
  • PBMCs were cultured in AIM-V Peiji (Gibco, #0870112) + 2% human AB serum (Gemini, #100-512), and recombinant human IL-2 (500 U/ml) was added (Shanghai Huaxin Biotech) Ltd.), activated T cells by adding CD3/CD28 magnetic beads (Invitrogen, #21013) in a ratio of 1:1 (cell: magnetic beads), and infected 48 hours later.
  • Retronectin (Takara, #T100A) was coated at 4 ° C overnight at a concentration of 5 ⁇ g/ml.
  • the activated T cells were added to the Retronectin-coated plate at a density of 1 ⁇ 10 6 cells/ml, and the high-purity virus collected in Step 2 was added (MOI ⁇ 10), centrifuged at 1800 rpm, 32° C. for 40 min, and centrifuged. After being cultured for 48 hours in an incubator, the solution was changed (AIM-V Petri + 2% human AB serum, IL-2 500 U/ml), and the beads were demagnetized, and then the cells were densified at a concentration of 5 ⁇ 10 5 cells/ml. After 6-10 days of culture, T cells infected with the hu8E3-28Z virus, that is, T cells expressing 8E3-28Z (SEQ ID NO: 48) were obtained.
  • the hu8E3-28Z T cells prepared in Example 8 were tested for killing of 3T3-mFAP and H1080-huFAP ⁇ cells by the following methods:
  • the 3T3-mFAP monoclonal cells were digested and counted, and T cells expressing 8E3-28Z CAR were counted by centrifugation.
  • 3T3-mFAP cells were used as target cells, and the effector cells were T cells expressing 8E3-28Z CAR.
  • the target ratios were 3:1, 1:1, 1:3, and the number of target cells was 10000/well, depending on the target.
  • a different number of effector cells ie 30000/well, 10000/well, 3333/well
  • Each group has 5 duplicate holes. After 18 hours of incubation, the LDH content in the supernatant was determined by CytoTox96 non-radioactive cytotoxicity kit (Promega, #G1780), and its killing activity was calculated.
  • T cells expressing 8E3-28Z CAR were able to specifically kill 3T3-mFAP cells expressing murine FAPa and H1080-huFAP ⁇ cells expressing human FAPa.

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Abstract

特异性结合FAPα的结合单元，编码此类结合单元的多核苷酸，包含此类多核苷酸的载体和宿主细胞。用于生成该抗原结合单元的方法和使用该FAPα特异性结合单元治疗疾病的方法。该特异性结合FAPα的结合单元能够有效结合表达FAPα的肿瘤细胞，而包含该结合单元的免疫效应细胞对表达FAP的肿瘤细胞表现出显著的杀伤能力。

Description

靶向成纤维激活蛋白α的结合单元及其应用 技术领域
本发明涉及生物医药领域；具体地说，本发明涉及特异性结合FAPα的结合单元，编码此类结合单元的多核苷酸，包含此类多核苷酸的载体和宿主细胞。本发明还涉及用于生成该抗原结合单元的方法和在疾病的治疗中使用它的方法。
背景技术
成纤维细胞激活蛋白α(Fibroblast activation proteinα,FAPα)是特异性表达于肿瘤相关成纤维细胞(CAF)表面的一种抗原分子(NCBI Reference Sequence:NP_001278736.1)，属于丝氨酸蛋白酶家族，具有胶原酶和二肽基肽酶活性，在肿瘤宿主界面基质的降解和重建中发挥着重要的作用，参与肿瘤的生长、浸润和转移，FAPα选择性表达于90％以上的恶性上皮肿瘤(如乳腺癌、卵巢癌、肺癌、结肠癌、胰腺癌、皮肤黑色素瘤、肾癌、膀胱癌等)的基质，胚胎组织、愈合创面及生理性重建的器官中，而在正常成体组织中一般不表达。这些特点使FAPα成为多种肿瘤的成像、诊断以及治疗的抗原靶标。
人FAPα最初是使用单克隆抗体(mAb)F19(记载于WO93/05804，ATCC编号HB8269)在培养的成纤维细胞中鉴定的。基于F19抗体的人源化开发了西罗珠单抗(Sibrotuzumab)/BIBH1，一项利用西罗珠单抗进行的I期研究证明了 131I标记的抗体的特异性肿瘤积累(Scott et al.,Clin Cancer Res 9,1639-1647(2003))。然而，未偶联的西罗珠单抗在具有转移性结肠直肠癌的患者中的一项早期II期试验因抗体缺少抑制肿瘤发展的功效而停止(Hofheinz et al.,Inkologie 26,44-48(2003))。
因此，本领域仍然需要增强的治疗方法，包括靶向FAPα、具有改良功效的抗体来治疗癌症。
发明内容
本发明目的在于发现特异性结合FAPα的结合单元，包括但不限于抗体以及开发靶向FAPα的免疫效应细胞。
在第一方面，本发明提供一种抗原结合单元，所述抗原结合单元包含轻链CDR区和重链CDR区，所述的重链CDR包含HCDR1，HCDR2，和HCDR3；所述的轻链CDR区包含LCDR1，LCDR2，和LCDR3；
其中，所述的HCDR1，HCDR2和HCDR3的序列独立选自下组：SEQ ID NO:1-3、7和8，所述的LCDR1，LCDR2和LCDR3的序列独立选自下组：SEQ ID NO:4-6、9和10。
在具体的实施方式中，所述的HCDR1具有SEQ ID NO:1或7所示的序列，所述的HCDR2具有SEQ ID NO:2或8所示的序列，所述的HCDR3具有SEQ ID NO:3所示的序列。
在具体的实施方式中，所述的HCDR1，HCDR2和HCDR3的序列选自以下任一组：
A.SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3；
B.SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:3。
在具体的实施方式中，所述的LCDR1具有SEQ ID NO:4或9所示的序列，所述的LCDR2具有SEQ ID NO:5或10所示的序列，所述的LCDR3具有SEQ ID NO:6所示的序列。
在具体的实施方式中，所述的LCDR1，LCDR2和LCDR3的序列选自以下任一组：
A.SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6；
B.SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:6。
在具体的实施方式中，所述的HCDR1具有SEQ ID NO:1或7所示的序列，所述的HCDR2具有SEQ ID NO:2或8所示的序列，所述的HCDR3具有SEQ ID NO:3所示的序列，所述的LCDR1具有SEQ ID NO:4或9所示的序列，所述的LCDR2具有SEQ ID NO:5或10所示的序列，所述的LCDR3具有SEQ ID NO:6所示的序列。
在具体的实施方式中，所述的HCDR1含有SEQ ID NO:1所示的序列、所述的HCDR2含有SEQ ID NO:2所示的序列、所述的HCDR3含有SEQ ID NO:3所示的序列、所述的LCDR1含有SEQ ID NO:4所示的序列、所述的LCDR2含有SEQ ID NO:5所示的序列、所述的LCDR3含有SEQ ID NO:6所示的序列；或者
所述的HCDR1含有SEQ ID NO:7所示的序列、所述的HCDR2含有SEQ ID NO:8所示的序列、所述的HCDR3含有SEQ ID NO:3所示的序列、所述的LCDR1含有SEQ ID NO:4所示的序列、所述的LCDR2含有SEQ ID NO:5所示的序列、所述的LCDR3含有SEQ ID NO:6所示的序列；或者
所述的HCDR1含有SEQ ID NO:1所示的序列、所述的HCDR2含有SEQ ID NO:2所示的序列、所述的HCDR3含有SEQ ID NO:3所示的序列、所述的LCDR1含有SEQ ID NO:9所示的序列、所述的LCDR2含有SEQ ID NO:10所示的序列、所述的LCDR3含有SEQ ID NO:6所示的序列。
在具体的实施方式中，所述的抗原结合单元具有SEQ ID NO:11或15所示的重链可变区，和SEQ ID NO:13或17所示的轻链可变区。
在优选的实施方式中，所述抗原结合单元是抗体，优选单克隆抗体或其活性片段。
在第二方面，本发明提供第一方面所述的抗原结合单元的突变体，所述突变体与第一方面所述的抗原结合单元竞争结合FAPα。
在具体的实施方式中，所述突变体与第一方面所述的抗原结合单元在轻链可变区序列上有90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的相同性；或者，所述突变体与权利要求1-8任一所述的抗原结合单元在在重链可变区序列上有90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的相同性；
优选地，所述突变体与第一方面所述的抗原结合单元在轻链CDR区，例如轻链CDR1或CDR2的序列上有70％以上；优选地，75％以上；更优选地，80％以上的相同性；或者， 所述突变体与第一方面所述的抗原结合单元在重链CDR区，例如重链CDR1或CDR2的序列上有60％以上；优选地，70％以上；更优选地，80％以上的相同性。
在具体的实施方式中，所述突变体与第一方面所述的抗原结合单元竞争结合FAPα上同一结合表位。
在第三方面，本发明提供一种抗原结合单元，所述抗原结合单元包含轻链CDR区和重链CDR区，所述的重链CDR包含HCDR1，HCDR2和HCDR3；所述的轻链CDR区包含LCDR1，LCDR2和LCDR3，其中，所述的HCDR1，HCDR2和HCDR3独立选自与SEQ ID NO:1-3、7、8中任一序列至少80％、优选至少85％、更优选至少90％、更优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％相似性的序列，所述的LCDR1，LCDR2和LCDR3独立选自与SEQ ID NO:4-6、9、10中任一序列至少80％、优选至少85％、更优选至少90％、更优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％相似性的序列。
在具体的实施方式中，所述的HCDR1具有SEQ ID NO:1或7所示的序列，所述的HCDR2具有SEQ ID NO:2或8所示的序列，所述的HCDR3具有SEQ ID NO:3所示的序列。
在具体的实施方式中，所述的HCDR1，HCDR2和HCDR3的序列选自以下任一组：
A.SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3；
B.SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:3。
在具体的实施方式中，所述的LCDR1具有SEQ ID NO:4或9所示的序列，所述的LCDR2具有SEQ ID NO:5或10所示的序列，所述的LCDR3具有SEQ ID NO:6所示的序列。
在具体的实施方式中，所述的LCDR1，LCDR2和LCDR3的序列选自以下任一组：
A.SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6；
B.SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:6。
在具体的实施方式中，所述的HCDR1具有SEQ ID NO:1或7所示的序列，所述的HCDR2具有SEQ ID NO:2或8所示的序列，所述的HCDR3具有SEQ ID NO:3所示的序列，所述的LCDR1具有SEQ ID NO:4或9所示的序列，所述的LCDR2具有SEQ ID NO:5或10所示的序列，所述的LCDR3具有SEQ ID NO:6所示的序列。
在具体的实施方式中，所述的HCDR1含有SEQ ID NO:1所示的序列、所述的HCDR2含有SEQ ID NO:2所示的序列、所述的HCDR3含有SEQ ID NO:3所示的序列、所述的LCDR1含有SEQ ID NO:4所示的序列、所述的LCDR2含有SEQ ID NO:5所示的序列、所述的LCDR3含有SEQ ID NO:6所示的序列；或者
所述的HCDR1含有SEQ ID NO:7所示的序列、所述的HCDR2含有SEQ ID NO:8所示的序列、所述的HCDR3含有SEQ ID NO:3所示的序列、所述的LCDR1含有SEQ ID NO:4所示的序列、所述的LCDR2含有SEQ ID NO:5所示的序列、所述的LCDR3含有SEQ ID NO:6所示的序列；或者
所述的HCDR1含有SEQ ID NO:1所示的序列、所述的HCDR2含有SEQ ID NO:2所示的序列、所述的HCDR3含有SEQ ID NO:3所示的序列、所述的LCDR1含有SEQ ID NO:9所示的序列、所述的LCDR2含有SEQ ID NO:10所示的序列、所述的LCDR3含有SEQ ID NO:6所示的序列。
在第四方面，本发明提供一种抗原结合单元，所述的抗原结合单元具有与SEQ ID NO:11或15所示的重链可变区的序列具有至少80％、优选至少85％、更优选至少90％、更优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％相似性的序列，和与SEQ ID NO:13或17所示的轻链可变区具有至少80％、优选至少85％、更优选至少90％、更优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％相似性的序列。
在第五方面，本发明提供第一到第四方面所述的抗原结合单元或抗原结合单元突变体，所述的抗原结合单元是单克隆抗体、全人抗体、人源化抗体、嵌合抗体。
在具体的实施方式中，所述的抗原结合单元是scFv、Fv、Fab、(Fab)2或者单域抗体。
在第六方面，本发明提供编码第一到第五方面所述的抗原结合单元的核酸。
在第七方面，本发明提供一种表达载体，其包含第六方面所述的核酸。
在第八方面，本发明提供一种宿主细胞，其包含第七方面所述的表达载体或基因组中整合有第六方面所述的核酸。
在第九方面，本发明提供一种双价蛋白，由第五方面所述的scFv序列与一个或多个重链恒定区域融合所形成具有人免疫球蛋白Fc区的抗体；
优选地，所述的人免疫球蛋白Fc区连接有缀合物；优选的，所述缀合物优选自荧光染料、细胞毒素、放射性同位素。
在第十方面，本发明提供一种多功能免疫辍合物，所述的多功能免疫辍合物包括:
第一到第五方面所述的抗原结合单元，以及与之连接的功能性分子；其中，所述的功能性分子选自:
(a)靶向肿瘤表面标志物的分子，包括结合免疫细胞表面标志物的抗体或配体；较佳地，所述的免疫细胞表面标志物包括:CD3，CD16，CD28；更佳地，所述的结合免疫细胞表面标志物的抗体是抗CD3抗体；最佳地，所述的靶向免疫细胞的表面标志物的分子是结合T细胞表面标志物的抗体；
(b)抑制肿瘤的分子,包括抗肿瘤的细胞因子或抗肿瘤的毒素，较佳的，所述的细胞因子包括:IL-12、IL-15、I型干扰素、TNF-alpha；
(c)靶向免疫细胞的表面标志物的分子或可检测标记物。
在第十一方面，本发明提供一种嵌合抗原受体，所述嵌合抗原受体的胞外域包含第一到第五方面所述的抗原结合单元，该抗原结合单元优选单链抗体或单域抗体。
在优选的实施方式中，所述嵌合抗原受体还包含跨膜域及胞内信号域，
所述的跨膜域，较佳地，选自TCR的α、β、zeta链，CD3ε，CD3ζ，CD4，CD5，CD8α，CD9，CD16，CD22，CD27，CD28，CD33，CD37，CD45，CD64，CD80，CD86，CD134，CD137，CD152，CD154，和PD1的跨膜区；更佳地，选自CD8α，CD4，CD45，PD1，CD154和CD28的跨膜域；
和/或
所述胞内信号域包含一个或多个共刺激信号域和/或初级信号域；其中，所述的共刺激信号域，较佳地，选自CARD11，CD2，CD7，CD27，CD28，CD30，CD40，CD54，CD83，OX40，CD137，CD134，CD150，CD152，CD223，CD270，PD-L2，PD-L1，CD278，DAP10，LAT，NKD2C SLP76，TRIM，FcεRIγ，MyD88，和41BBL的胞内信号区；和/或所述初级信号域选自TCRξ、FcRγ、FcRβ、CD3γ、CD3δ、CD3ε，CD5，CD22，CD79a，CD79b，CD278(也称作“ICOS”)和CD66d，和CD3ζ；更佳地，选自CD137，CD134，CD28和OX40；和/或所述初级信号域选自CD3ζ。
在优选的实施方式中，所述的跨膜域选自CD8α或CD28，所述共刺激信号域选自CD137或CD28的胞内信号域，所述初级信号域选自CD3ζ。
在具体的实施方式中，所述的嵌合抗原受体包括如下顺序连接的抗体，跨膜区和胞内信号区：
第一到第五方面所述的抗原结合单元、CD8和CD3ζ；
第一到第五方面所述的抗原结合单元、CD8、CD137和CD3ζ；
第一到第五方面所述的抗原结合单元、CD28分子的跨膜区、CD28分子的胞内信号区和CD3ζ；或
第一到第五方面所述的抗原结合单元、CD28分子的跨膜区、CD28分子的胞内信号区、CD137和CD3ζ。
在第十二方面，本发明提供编码第十一方面所述的嵌合抗原受体的核苷酸序列。
在第十三方面，本发明提供一种表达载体，所述表达载体包含第十二方面所述的核苷酸序列。
在第十四方面，本发明提供一种病毒，所述的病毒包含第十三方面所述的表达载体。
在第十五方面，本发明提供一种基因修饰的免疫细胞，其转导有第十二方面所述的核 苷酸序列、或第十三方面所述的表达载体、或第十四方面所述的病毒；或者，其表达第十一方面所述的嵌合抗原受体。
在优选的实施方式中，所述的免疫细胞包括但不限于以下细胞及其组合：T淋巴细胞，NK细胞或NKT细胞。
在具体的实施方式中，所述基因修饰的免疫细胞还表达有除嵌合抗原受体之外的其他序列，所述其他序列包括细胞因子、另一种嵌合抗原受体、趋化因子受体、降低PD-1表达的siRNA或者阻断PD-L1的蛋白、TCR、或安全开关；
较佳地，所述的细胞因子包括IL-12、IL-15、IL-21、或I型干扰素；
较佳地，所述趋化因子受体包括CCR2、CCR5、CXCR2、或CXCR4；
较佳地，所述安全开关包括iCaspase-9、Truancated EGFR或RQR8。
在第十六方面，本发明提供第一到第五方面所述的抗原结合单元、或第九方面所述的双价蛋白、或第十方面所述的免疫辍合物、或第十一方面所述的嵌合抗原受体、或第十二方面所述的核苷酸序列、或第十三方面所述的表达载体、或第十四方面所述的病毒、或第十五方面所述的免疫细胞的用途，用于制备治疗、预防或诊断高表达FAPα相关的疾病的药物或试剂。
在具体的实施方式中，所述的高表达FAPα相关的疾病是肿瘤。
在具体的实施方式中，所述的肿瘤是乳腺癌、卵巢癌、肺癌、结肠癌、胰腺癌、皮肤黑色素瘤、肾癌、膀胱癌。
在第十七方面，本发明提供一种药物组合物，所述药物组合物包含第一到第五方面所述的抗原结合单元或编码该抗原结合单元的核酸；或
第九方面所述的双价蛋白；或
第十方面所述的免疫辍合物或编码该辍合物的核酸；或
第十一方面所述的嵌合抗原受体或编码该嵌合抗原受体的核酸；或
第十五方面所述的基因修饰的免疫细胞。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了SDS PAGE分析镍柱纯化重组human FAPα_His(huFAP),mouse FAP_His(mFAP)的结果；
图2显示了ELISA测定噬菌体克隆表达上清液和重组huFAPa，mFAP的结合；
图3显示了FACs检测部分纯化的克隆和U-87MG以及A549细胞的结合；
图4显示了Biacore测定1A7、8E3(scFv)结合huFAP，mFAP的亲和力；
图5显示了抗体1A7、8E3与亲本抗体10A4的序列比对(scFv)；
图6为SDS PAGE分析纯化的抗体1A7、8E3(scFv_Fc)；
图7为Western blot检测3T3-mFAP，HT1080-huFAPα稳转细胞系的结果；
图8为FACs检测3T3-mFAP(a)，HT1080-huFAPα(b)稳转细胞系的结果；
图9显示了FACs测定抗体10A4、1A7、8E3(scFv_Fc)与U-87MG、3T3-mFAP、HT1080-huFAPα细胞的结合；
图10是利用Anti-Flag抗体FACs检测3T3-mFAP(a)、HT1080-huFAP(b)稳转细胞系中mFAPα及huFAPα的表达；
图11是FACs检测hu8E3-28Z CAR在T细胞中的表达；
图12是细胞毒性检测表达hu8E3-28Z CAR的T细胞对3T3-mFAP(a)及H1080-huFAPα(b)细胞的杀伤。
具体实施方式
发明人经过广泛而深入的研究，出乎意料地发现特异性结合FAP的抗体，在此基础上开发了特异性靶向FAP的免疫效应细胞。本发明的抗体能够有效结合表达FAP的肿瘤细胞，本发明的免疫效应细胞对表达FAP的肿瘤细胞表现出显著的杀伤能力，从而能够有效而安全地应用于多发性骨髓瘤等恶性肿瘤的治疗。在此基础上完成了本发明。
以下具体说明详尽地展示了本文所公开的实施方案。应当理解，本说明书并非意欲仅限于此处所公开的具体的实施方案，而是可以发生改变。本领域技术人员将理解，本说明书中所公开的内容可以有多种改变或变化，而均涵盖于所公开的范围和原则之内。除非另有说明，每个实施方案均可与任何其他实施方案任意组合。
本文所公开的某些实施方案包含了数值范围，并且本发明的某些方面可采用范围的方式描述。除非另有说明，应当理解数值范围或者以范围描述的方式仅是出于简洁、便利的目的，并不应当认为是对本发明的范围的严格限定。因此，采用范围方式的描述应当被认为具体地公开了所有可能的子范围以及在该范围内的所有可能的具体数值点，正如这些子范围和数值点在本文中已经明确写出。
当涉及可测量值比如量、暂时持续时间等时，术语“约”是指包括指定值的±20％、或在某些情况下±10％、或在某些情况下±5％、或在某些情况下±1％、或在某些情况下±0.1％的变化。
术语定义
本文所用的术语具有与本领域技术人员常规理解相似的含义。为清晰起见，现对其中一些术语定义如下。
本文所用的术语“FAPα”是指成纤维细胞激活蛋白α。人成纤维细胞激活蛋白α由760 个氨基酸残基组成的跨膜蛋白(NCBI Reference Sequence:NP_001278736.1)。如果没有特别说明，本文中的FAPα是指人成纤维细胞激活蛋白α。鼠的成纤维细胞激活蛋白由761个氨基酸残基组成(由760个氨基酸残基组成的跨膜蛋白，NCBI Reference Sequence:NP_032012.1)。
本文所用的术语“结合单元”、“抗原结合单元”或“抗原结合蛋白”具有相同的含义，在本文可以互换使用，是指免疫球蛋白分子和免疫球蛋白分子的任何形式的免疫活性部分；即，包含特异性结合抗原或与抗原免疫反应的抗原结合位点的分子。结构上，最简单的天然产生的抗体(例如，IgG)具有四个多肽链，通过二硫键相互连接的两个重(H)链和两个轻(L)链。免疫球蛋白代表了包括几种分子类型在内的一大族分子，例如IgD、IgG、IgA、IgM和IgE。抗原结合单元包括但不限于Fv、scFv、dFv、dAb、二链抗体、三链抗体、四链抗体、结构域Ab、Fab片段、Fab’、(Fab’) 2、双特异性抗体和多特异性的抗体。
本文所用的术语“竞争结合”是指两种或多种物质与结合对象的同一位置，例如，某抗原的同一结合表位相结合；换言之，结合在同一结合对象上的不同位置的不同物质不存在本文所述的“竞争结合”的关系。
本文所用的术语“抗原结合单元”还包括多物种来源的免疫球蛋白分子，包括无脊椎动物和脊椎动物。应用于抗原结合单元的术语“人”是指由人类基因或其片段表达的免疫球蛋白分子。应用于非人(例如啮齿动物或灵长类动物)抗体的术语“人源化”是包含源自非人免疫球蛋白的最小序列的杂交免疫球蛋白、免疫球蛋白链或其片段。对于其大部分，人源化抗体是人免疫球蛋白(受者抗体(recipient antibody))，其中来自受者互补决定区(CDR)的残基被来自具有所需特异性、亲和力和容量的非人物种(如小鼠、大鼠、兔或灵长动物)的CDR(供者抗体)的残基取代。在某些情况中，非人免疫球蛋白的Fv框架区(FR)残基被相应的人残基取代。另外，人源化抗体可以包含既不在受者抗体也不在输入的CDR或框架序列中发现的残基。进行这些修饰以进一步完善和优化抗体性能和在引入人体时最大程度降低免疫原性。一般而言，人源化抗体包含基本上全部的至少一个和通常两个可变域，其中全部或基本上全部CDR区域对应于非人免疫球蛋白的CDR区域且全部或基本上全部FR区域为非人免疫球蛋白序列的FR区域。
本文所用的术语“抗体”，作为本领域已知的那些术语，指免疫系统的抗原结合蛋白。如本文提到的术语“抗体”包括具有抗原结合区域的完整的全长抗体及其中“抗原结合部分”或“抗原结合区域”保留的其任何片段、或其单链例如单链可变片段(scFv)。天然抗体指包含通过二硫键互联的至少两条重(H)链和两条轻(L)链或其抗原结合片段的糖蛋白。术语“抗体”还包括抗体(特别是本文所述抗体)的所有重组形式，例如在原核细胞中表达的抗体，未糖基化的抗体以及与抗原结合的抗体片段和下文所诉的衍生物。每条重链由重链可变区(本文缩写为V H)和重链恒定区组成。每条轻链由轻链可变区(本文缩写为V L)和轻链恒定区组成。V H和V L可进一步细分为称为互补性决定区(CDR)的高变区，它们散布在称为构架区(FR)的更保守区域中。每条V H和V L由三个CDR和四个FR组成，从氨基端至羧基端按以下顺序排列：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。重链和轻链的可变区含有与抗原相互作用的结合结构域。抗体的恒定区可介导该免疫球蛋白与宿主组织或因子的结合，所述宿主组织 或因子包括免疫系统的多种细胞(如效应细胞)和经典补体系统的第一成分(C1q)。
本文所用的术语“Fab”或“Fab区”包括包含V H、C H1、V L和C L免疫球蛋白结构域的多肽。Fab可以指分离的该区域，或者位于全长抗体或抗体片段环境中的该区域。
本文中的术语“Fc”或“Fc区”包括包含除第一恒定区免疫球蛋白结构域以外的抗体恒定区的多肽。因而，Fc指IgA、IgD和IgG的最后两个恒定区免疫球蛋白结构域，和IgE和IgM的最后三个恒定区免疫球蛋白结构域，和这些结构域N端的柔性铰链。对于IgA和IgM，Fc可包括J链。对于IgG，Fc包括免疫球蛋白结构域Cγ2和Cγ3和在Cγ1和Cγ2之间的铰链。虽然Fc区的边界可以改变，但人IgG重链Fc区通常定义为在其羧基端包含残基C226或P230，其中编号是根据Kabat的EU索引。对于人IgG1，Fc在本文定义为包含残基P232至其羧基端，其中编号是根据Kabat中的EU索引。Fc可以指分离的该区域，或者位于Fc多肽，例如抗体，环境中的该区域。
本文中使用的术语“亲本抗体”或“亲本免疫球蛋白”包括未修饰的抗体，所述抗体之后经修饰产生变体。所述亲本抗体可以使天然存在的抗体，或者天然存在的抗体的变体或改造版本。亲本抗体可以指抗体本身，包含所述亲本抗体的组合物，或其编码氨基酸序列。本文中使用的术语“亲本抗体”或“亲本免疫球蛋白”包括之后经修饰产生人源化抗体的鼠抗体或嵌合抗体。
本文中使用的术语“变体抗体”或“抗体变体”或“变体”包括由于相比亲本的至少一个氨基酸修饰，而不同于亲本抗体序列的抗体序列。抗体变体可以指抗体本身，也可以指包含所述亲本抗体的组合物。在具体的实施方式指，本文所述的变体抗体的轻链可变区序列与亲本抗体的轻链可变区序列有90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的相同性；本文所述的变体抗体的重链可变区序列与亲本抗体的重链可变区序列有90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的相同性。在优选的实施方式中，本文所述的变体抗体的轻链CDR区，例如轻链CDR1或CDR2的序列与亲本抗体的轻链CDR区，例如轻链CDR1或CDR2序列有70％以上的相同性；优选地，75％以上的相同性；更优选地，80％以上的相同性；本文所述的变体抗体的重链CDR区，例如重链CDR1或CDR2的序列与亲本抗体的重链CDR区，例如重链CDR1或CDR2序列有60％以上的相同性；优选地，70％以上的相同性；更优选地，80％以上的相同性。
术语“氨基酸修饰”包括氨基酸取代、添加和/或缺失，“氨基酸取代”意指用另一种氨基酸替换亲本多肽序列中特定位置上的氨基酸。例如，取代R94K指94位的精氨酸被赖氨酸替换，本文中使用的“氨基酸插入”意指在亲本多肽序列中的特定位置添加氨基酸。文中使用的“氨基酸缺失”或“缺失”意指去除亲本多肽序列中特定位置上的氨基酸。
本文中使用的术语“保守修饰”或“保守序列修饰”意指不显著影响或改变含有所述氨基酸序列的抗体的结合特征的氨基酸修饰。此类保守修饰包括氨基酸取代、插入和缺失。可通过本领域已知的标准技术将修饰导入本发明的抗体中，例如定点诱变和PCR介导的诱变。保守的氨基酸取代是用具有相似侧链的氨基酸残基替换氨基酸残基的取代。本领域已经定义了具有相似侧链的氨基酸残基家族。这些家族包括含碱性侧链的氨基酸(例如，赖氨酸、精 氨酸、组氨酸)、酸性侧链(例如，天冬氨酸、谷氨酸)、不带电的极性侧链(例如，甘氨酸、天冬酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如，丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)、β分支侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳香族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)。因而，可以用其他相同侧链家族的氨基酸残基替换本发明抗体的CDR区中或框架区中的一个或多个氨基酸残基，并可以测试所改变的抗体(变体抗体)保留的功能。
抗体片段包括但不限于：(i)由V L、V H、C L和C H1结构域组成的Fab片段，包括Fab’和Fab’-SH，(ii)V H和C H1结构域组成的Fd片段，(iii)由单个抗体的V L和V H结构域组成的Fv片段；(iv)由单个可变区组成的dAb片段(Ward等，1989，Nature 341：544-546)；(v)F(ab’) 2片段，包含2个连接的Fab片段的二价片段；(vi)单链Fv分子抗原结合位点(Bird等，1988，Science 242：423-426；Huston等，1988，Proc.Natl.Acad.Sci.U.S.A 85:5879-5883)；(vii)双特异性单链Fv二聚体(PCT/US92/09965)；(viii)“二体”或“三体”，通过基因融合构建的多价或多特异性片段(Tomlinson等，2000，Methods Enzymol.326:461-479；WO94/13804；Holliger等，1993,Proc.Natl.Acad.Sci.U.S.A 90:6444-6448)；和(ix)与相同或不同抗体遗传融合的scFv(Coloma&Morrison,1997,Nature Biotechnology 15,159-163)。
根据恒定区基因测定，将抗体分类，也称为同种型。人的恒定轻链分为К(CК)和λ(Cλ)轻链。重链分为μ、δ、γ、α或ε，并分别定义抗体的同种型IgM、IgD、IgG、IgA和IgE。IgG类是最常用于治疗目的的。在人中，该类别包括亚类IgG1、IgG2、IgG3和IgG4。在小鼠中，该类别包括亚类IgG1、IgG2a、IgG2b和IgG3。IgM具有亚类，包括但不限于IgM1和IgM2。IgA具有若干亚类，包括但不限于IgA1和IgA2。因而，本文中使用的“同种型”意指通过恒定区的化学和抗原特征定义的免疫球蛋白的任何类或亚类。已知的人免疫球蛋白同种型是IgG1、IgG2、IgG3、IgG4、IgA1、IgA2、IgM1、IgM2、IgD和IgE。
抗FAPα的抗体
在本公开中，描述了具有基于scFv的抗原结合区域的抗原结合蛋白，包括抗体。其中使用重组FAPα，从人scFv噬菌体展示文库选择scFv。
在一些实施方案中，本发明包括具有scFv序列的抗体，所述scFv序列与一个或多个重链恒定区域融合以形成具有人免疫球蛋白Fc区的抗体以产生双价蛋白，从而增加抗体的总体亲和力和稳定性。此外，Fc部分允许将其他分子(包括但不限于荧光染料、细胞毒素、放射性同位素等)与例如用于抗原定量研究中的抗体直接缀合，以便固定抗体用于亲和力测量、用于定向递送治疗药、使用免疫效应细胞测试Fc介导的细胞毒性和许多其它应用。
本文提供的结果突出表现出本发明抗体在靶向FAPα时的特异性、灵敏性和效用。
本发明的分子基于使用噬菌体展示鉴定和选择单链可变片段(scFv)，所述单链可变片段的氨基酸序列赋予分子针对FAPα的特异性并且形成本发明的全部抗原结合蛋白的基础。因此，所述scFv可以用来设计一系列不同“抗体”分子，包括例如全长抗体、其片段如Fab和F(ab’) 2、融合蛋白(包括scFv_Fc)、多价抗体、即，具有针对相同抗原或不同抗原的多于一种 特异性的抗体，例如，双特异性T细胞结合抗体(BiTE)、三抗体等(见Cuesta等，Multivalent antibodies:when design surpasses evolution,Trends in Biotechnology 28:355-362,2010)。
在抗原结合蛋白是全长抗体的一个实施方案中，本发明抗体的重链和轻链可以是全长(例如，抗体可以包括至少一条并优选地两条完整重链，和至少一条并优选地两条完整轻链)或可以包括抗原结合部分(Fab、F(ab’) 2、Fv或scFv)。在其他实施方案中，抗体重链恒定区选自例如IgG1、IgG2、IgG3、IgG4、IgM、IgA1、IgA2、IgD和IgE。抗体类型的选择将取决于所设计的抗体欲引发的免疫效应子功能。在构建重组免疫球蛋白时，各种免疫球蛋白同种型的恒定区的适宜氨基酸序列和用于产生广泛种类抗体的方法是本领域技术人员已知的。
在另一方面，本发明提供了结合FAPα的抗原结合单元，其包括选自SEQ ID NO:11、15的重链可变区序列。
在另一方面，本发明提供了结合FAPα的抗原结合单元，其包括选自SEQ ID NO:13、17的轻链可变区序列。
考虑到这些重链和轻链可变区序列各自可以结合FAPα，可以“混合和匹配”重链和轻链可变区序列来产生本发明的抗FAPα的结合分子。
在另一个方面，本发明提供了结合FAPα的抗体或其片段的变体。因而本发明提供了抗体或其片段，具有与本发明抗体的重链或轻链的可变区序列至少80％相同的重链和/或轻链可变区。优选的，重链和/或轻链可变区的氨基酸序列同一性是至少85％，更优选至少90％，最优选至少95％，特别是96％，更特别97％，甚至更特别98％，最特别99％，包括例如80％，81％，82％，83％，84％，85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％和100％。
抗FAPα的抗体特性
评估抗体，例如抗FAP的抗体的结合能力的标准测定是本领域已知的，包括例如ELISA、Biacore、Western印迹和流式细胞仪分析。合适的测定详细描述在实施例中。
核酸、载体和宿主细胞
本发明还提供了编码结合FAPα的抗体和其片段分离的核酸、载体以及包含所述核酸或载体的宿主细胞。核酸可位于完整细胞中、细胞裂解液中或者以部分纯化的或基本纯化的形式。
可以使用标准的分子生物学技术获得本发明的核酸，例如可以通过标准的PCR扩增或cDNA克隆技术，获得编码抗体的轻链和重链或者编码VH和VL区段的cDNA。对于从免疫球蛋白基因文库获得的抗体(例如，使用噬菌体展示技术)，可以从文库回收编码抗体的一种或多种核酸。向宿主细胞中导入外源核酸的方法是本领域普遍已知的，并可随所使用的宿主细胞而变化。
为了表达蛋白质，可以将编码本发明抗体的核酸整合到表达载体中。多种表达载体可用于蛋白质表达。表达载体可包括自我复制的染色体外载体，或整合到宿主基因组中的载体。 用于本发明的表达载体包括但不限于使蛋白质能够在哺乳动物细胞、细菌、昆虫细胞、酵母和体外系统中表达的那些。如本领域已知的，多种表达载体是可商业或其他方式获得的。可用于本发明中来表达抗体。
本发明的优点：
1.本发明提供了特异性结合FAPα的抗体；
2.本发明提供了特异性靶向FAPα的免疫效应细胞；和
3.本发明的抗体能够有效结合表达FAPα的肿瘤细胞，本发明的免疫效应细胞对表达FAP的肿瘤细胞表现出显著的杀伤能力。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社，2002中所述的条件，或按照制造厂商所建议的条件。
实施例1.FAPα重组蛋白的制备
体外合成人FAPα的胞外段Leu26-Asp760基因，在该基因C端插入His标签，中间以“GS”相连接，形成融合表达蛋白human FAPα_His(SEQ ID NO:19)，相对应的基因序列如SEQ ID NO:20所示。
体外合成鼠的FAP胞外段基因(Leu26-Asp761)C端插入His标签，中间以“GS”相连接，形成融合表达蛋白mouse FAPα_His(SEQ ID NO:21)，相对应的基因序列如SEQ ID NO:22所示。
采用293F细胞分别瞬时转染表达human FAPα_His和mouse FAPα_His，转染的方法参照293F细胞的使用说明书。转染后采用镍柱亲和纯化。
纯化后SDS-PAGE电泳，结果如图1所示，E2中都得到了纯度较高的目的蛋白。
用0.22um的膜过滤后，使用截流量为10KD的millipore超滤管进行浓缩，浓缩至体积1ml以内，使用PD-Midi脱盐柱脱盐，收集得到人和鼠的FAPα重组蛋白。
实施例2.使用全人噬菌体展示文库筛选针对FAP特异的scFv
本发明使用的噬菌体展示文库为本申请人构建的全人的天然的scFv噬菌体文库，库容为1E+11。利用本领域技术人员已知的筛选方法，包被10ug/ml抗原human FAPα于免疫管，将噬菌体文库加入包被了human FAPα的免疫管中结合1.5小时，筛选和富集与FAPα特异性结合的scFv噬菌体克隆。
通过针对FAPα的标准ELISA方法确定阳性克隆，共筛选了1344个克隆，挑取其中ELISA结合信号最高的96个克隆(图2)，经测序，得到16个单一的序列，表达纯化这16个克隆并通过Biacore测定亲和力。结果如表1所示：
表1
Ligand ka(1/Ms) kd(1/s) KD(M) Rmax(RU) Chi 2(RU 2) Model
12A6 1.39E+05 4.55E-03 3.28E-08 17.1 2.69 1:1 Binding
8B8 3.76E+04 2.88E-03 7.64E-08 82.9 0.889 1:1 Binding
8B1 4.19E+04 3.39E-03 8.08E-08 39.7 1.33 1:1 Binding
10B9 5.08E+04 5.92E-03 1.17E-07 26.2 0.949 1:1 Binding
8B2 5.49E+04 6.60E-03 1.20E-07 39.4 0.652 1:1 Binding
12G12 5.93E+04 7.60E-03 1.28E-07 19.5 0.667 1:1 Binding
10C9 3.27E+04 4.26E-03 1.30E-07 22.6 1.27 1:1 Binding
8C4 2.74E+04 3.71E-03 1.35E-07 87.7 0.971 1:1 Binding
10A3 3.18E+04 5.14E-03 1.62E-07 48.2 1.02 1:1 Binding
10H7 3.84E+04 6.46E-03 1.68E-07 37.4 0.573 1:1 Binding
8E3 3.56E+04 6.21E-03 1.74E-07 42.1 1 1:1 Binding
10A4 1.94E+04 3.81E-03 1.96E-07 47 1.63 1:1 Binding
8A3 1.98E+04 6.76E-03 3.42E-07 112.4 1.74 1:1 Binding
12E11 1.58E+04 6.36E-03 4.03E-07 65.2 1.15 1:1 Binding
8A9 7.53E+03 6.12E-03 8.13E-07 131.3 0.761 1:1 Binding
FACs检测和内源性表达FAPα的U-87MG细胞(购自中国科学院细胞库)结合，不表达FAPα的A549细胞(购自中国科学院细胞库)做为阴性对照细胞。使用Guava easyCyteTM HT System仪器检测，结果如图3所示，抗体10A4特异性的结合U-87MG细胞。
经测序分析，10A4的重链可变区为SEQ ID NO:11所示的序列，重链的HCDR1、HCDR2、HCDR3的序列分别如SEQ ID NO:1、2、3所示。10A4的轻链可变区为SEQ ID NO:13所示的序列，轻链的LCDR1、LCDR2、LCDR3的序列分别如SEQ ID NO:4、5、6所示。
实施例4.抗体10A4的亲和力成熟文库的构建以及亲和力成熟文库的筛选
基于抗体10A4的亲和力成熟文库的构建保留了轻链以及重链的CDR3区域，通过简并引物，分别随机化轻链的CDR1和CDR2或者重链的CDR1和CDR2构建了两个亲和力成熟文库。具体构建方法简述如下：
首先基于抗体10A4的scFv(氨基酸序列SEQ ID NO:23，核苷酸序列SEQ ID NO:24)构建模板质粒。对于轻链CDR1和CDR2随机化的噬菌体文库，使用引物LMF(SEQ ID NO:41)和F10L1R(SEQ ID NO:45)PCR扩增片段1；使用引物F10L2F(SEQ ID NO:46)和FdR(SEQ ID NO:44)PCR扩增片段2；通过搭桥PCR连接片段1和片段2得到含有随机化序列的scFv全长，然后用NcoI和NotI酶切全长片段，通过T4连接酶连接入同样酶切的模板质粒中。并电转化至TG1感受态细胞中，库容为1.5E+9。对于重链CDR1和CDR2随机化的噬菌体文库，使用引物LMF(SEQ ID NO:41)和F10H1R(SEQ ID NO:42)，PCR扩增片段3；使用引物F10H2F(SEQ ID NO:43)和FdR(SEQ ID NO:44)，PCR扩增片段4；然后通过搭桥PCR连接片段3和片段4得到含有随机化序列的scFv全长，然后用NcoI和NotI酶切全长片段，通过T4连接酶连接入同样酶切的模板质粒中。并电转化至TG1感受态细胞中，库容为6.6E+8。
亲和力成熟文库的筛选与实施例2中的描述基本一致。包被5ug/ml或者1ug/ml抗原human FAPα于免疫管，将噬菌体文库加入包被了human FAPα的免疫管中结合1.5小时，经筛选和富集，得到与human FAPα特异性结合的scFv噬菌体克隆。
通过标准ELISA方法确定阳性克隆，挑取ELISA信号高的克隆表达纯化，经Biacore测定(见图4)，得到2个克隆(1A7、8E3)表现比亲本抗体10A4要好，亲本抗体10A4对人 FAPα的亲和力为197nM，如图4所示，抗体1A7对人FAPα的亲和力为13.5nM，相比10A4提高了14倍，抗体8E3对人FAPα的亲和力为81.6nM，相比10A4提高了2.4倍。两个抗体都与鼠FAP结合，亲和力分别为112nM和267nM。
对1A7、8E3进行测序，测序的结果显示，与亲本抗体10A4相比，抗体1A7一共有7处点突变，其中2处位于重链的CDR1，第31位由Ser变成Pro，第33位由Ala变成Thr；有5处位于重链的CDR2，第52位由Ile变成Val，第54位有Ile变成Asn，第55位由Phe变成Val，第57位由Thr变成Val，第59位由Asn变成Thr。抗体8E3一共有6处点突变，其中4处位于轻链的CDR1，第32位由Ser变成Pro，第35位由Tyr变成Phe，第37位由Tyr变成His，第39位由Asp变成Tyr；有两处位于轻链的CDR2，第55位由Leu变成Val，第57位由Ser变成Gly。
序列比对的结果见图5所示，抗体同一性的比较发现：1A7和10A4相比，重链的CDR1的序列具有60％的相似性，重链的CDR2具有70.5％的相似性，重链的CDR3具有100％的相似性，重链可变区具有93.9％的相似性(共116个氨基酸，7个不同)。8E3和10A4相比，轻链的CDR1的序列具有75％的相似性(共16个氨基酸，4个不同)，轻链的CDR2具有71.4％的相似性(共7个氨基酸，2个不同)，轻链的CDR3具有100％的相似性，轻链可变区具有94.6％的相似性(共113个氨基酸，6个不同)。
实施例5.scFv_Fc融合抗体的构建及其在真核细胞中的瞬转表达纯化
分别针对10A4、1A7、8E3的V H和V L片断设计引物，引入由15个柔性氨基酸(GGGGSGGGGSGGGGS)组成的linker连接组成scFv(10A4，SEQ ID NO:23；1A7，SEQ ID NO:25；8E3，SEQ ID NO:27)；在V H上游引入Nhe I的酶切位点和保护碱基，在V L的下游引入BamHI的酶切位点和保护碱基。1％琼脂糖凝胶电泳分析PCR产物并纯化回收。酶切后连接入含有人IgG1重链恒定区的Fc段Asp104-Lys330的V152(购自上海锐劲生物技术有限公司)真核表达载体中。采用293fectin TM Transfection reagent(Invitrogen,12347-019)或者聚乙烯亚胺(PEI)(Sigma-Aldrich,408727)瞬时转染对数生长期的293F细胞。转染5-7天后收集培养上清通过Protein A进行亲和纯化。SDS PAGE分析纯化产物，如图6所示，还原条件下，条带大小为50kD，符合预期。
实施例6.HT1080-huFAPα,3T3-mFAP稳转细胞系的构建
1.慢病毒包装质粒pWPT-huFAPα_Flag，pWPT-mFAP_Flag的构建
体外合成huFAPa的全长序列，并在C端插入Flag标签(SEQ ID NO:29)，并通过MluI/SalI双酶切，插入同样双酶切的慢病毒包装质粒pWPT中。体外合成mFAPa的全长序列，并在C端插入Flag标签(SEQ ID NO:30)，并通过MluI/SalI双酶切，插入同样双酶切的慢病毒包装质粒pWPT中。
2.mFAP病毒液和huFAPα病毒液的制备
接种6×10 6细胞至培养皿中，当细胞汇合度为70％-80％时进行转染，转染前1h把293T 的培养基更换为完全培养基。在EP管中加入800ul DMEM，随后加入5ug pWPT-mFAP-Flag质粒，7.5ug psPAX.2质粒，2.5ug pMD2.G质粒涡旋振荡8s混匀；然后在另一个EP管中，加入800ul DMEM和45uL 1ug/ul PEI室温下静置5分钟。将质粒混合液逐滴加入PEI孵育液中，混匀室温静置20分钟。然后将配置的质粒/PEI混合液逐滴加入细胞中。转染6小时候，将培养基更换为新鲜的完全培养基。转染72小时候，收集细胞培养上清，4度，3000rpm离心15分钟，取上清用0.45um滤膜过滤，得到mFAP病毒液。
huFAP病毒液的制备方法同mFAP病毒液的制备。
3.病毒感染细胞
第一天，接种1×10 5 3T3细胞于6cm培养皿。第二天，弃上清并加入1ml新鲜的完全培养基，然后加入4ml mFAP病毒液液和终浓度为6ug/ml的polybrene培养，得到3T3-huFAP混合克隆细胞。
采用huFAPα病毒感染HT1080细胞，方法同mFAP病毒液感染3T3细胞。
4. 3T3-mFAP、HT1080-huFAPα混合克隆Western blot鉴定
3T3-huFAP混合克隆细胞或者HT1080-huFAPα混合克隆细胞以蛋白裂解液裂解，离心后取上清进行BCA测定浓度，以3T3作为阴性对照，一抗：anti-FLAG-HRP(M2)，1:400稀释用。结果如图7所示。
5.流式检测
分别将HT1080-huFAPα和3T3-mFAP的混合克隆细胞消化后计数，以有限稀释法进行单克隆铺板，得到HT1080-huFAPα和3T3-mFAP的稳转细胞系。分别取4×105cells进行流式检测，使用Guava easyCyteTM HT System仪器检测。结果如图8所示，两株稳转细胞系中都能检测到FAP的表达。
采用Anti-Flag抗体流式检测3T3-mFAP，HT1080-huFAPα细胞系，取1×10 6个细胞进行流式检测，结果如图10所示，通过检测FAP上携带的Flag标签，在两株稳转细胞系中都能检测到FAP的表达。
实施例7.FACs测定抗体10A4、1A7、8E3的scFv_Fc与U-87MG、3T3-mFAP、HT1080-huFAPα细胞的结合
以3T3细胞和HT1080细胞做为阴性对照细胞。具体步骤为：收获细胞，用生长培养基洗涤细胞一次，重悬于PBS中，调整细胞浓度为4E+5细胞/ml。在冰上将200nM抗体与细胞育30分钟。其后与FITC标记的抗人IgG第二抗体孵育。两次洗涤之后，使用Guava easyCyteTM HT System仪器检测。
结果如图9所示，抗体10A4能结合稳定转染的huFAPα的HT1080细胞系，稳定转染mFAP的3T3细胞系以及内源性表达huFAPα的U-87MG细胞，但是不结合不表达huFAPα的HT1080细胞系，非特异性的结合3T3细胞系。抗体8E3和阴性细胞系3T3、HT1080都不结合，但是和FAP表达阳性的细胞系HT1080-huFAPα、3T3-mFAP、U-87MG都结合。抗体1A7和阴性细胞系3T3、HT1080都不结合，和HT1080-huFAPα、3T3-mFAP都结合，但 是不结合U-87MG细胞。
实施例8.靶向FAPα的嵌合型抗原受体(CAR)修饰的T细胞的制备
1.慢病毒包装质粒pRRL-hu8E3-28Z的构建
以PRRLSIN-cPPT.EF-1α为载体，构建了表达抗体10A4、1A7、8E3的二代嵌合抗原受体的慢病毒质粒，包括PRRLSIN-cPPT.EF-1α-10A4-28Z、PRRLSIN-cPPT.EF-1α-1A7-28Z以及PRRLSIN-cPPT.EF-1α-8E3-28Z。10A4-28Z序列由CD8α信号肽(SEQ ID NO:32)、10A4scFv(SEQ ID NO:24)、CD8hinge(SEQ ID NO:34)、CD28跨膜区(SEQ ID NO:36)和胞内信号传导结构域(SEQ ID NO:38)以及CD3的胞内段CD3ξ(SEQ ID NO:40)组成；1A7-28Z序列由CD8α信号肽(SEQ ID NO:32)、1A7scFv(SEQ ID NO:26)、CD8hinge(SEQ ID NO:34)、CD28跨膜区(SEQ ID NO:36)和胞内信号传导结构域(SEQ ID NO:38)以及CD3的胞内段CD3ξ(SEQ ID NO:40)组成；8E3-28BB序列由CD8α信号肽(SEQ ID NO:32)、8E3-scFv(SEQ ID NO:28)、CD8hinge(SEQ ID NO:34)、CD28跨膜区(SEQ ID NO:36)和胞内信号传导结构域(SEQ ID NO:38)以及CD3的胞内段CD3ξ(SEQ ID NO:40)组成。
2.质粒转染293T包装hu8E3-28Z慢病毒
a)取293T细胞消化后以1.25×10 7铺至15cm平皿，37℃培养至细胞汇合度为70％-80％，将上清换成10mL新鲜的10％FBS培基。
b)配制质粒/PEI混合液
取pRRL-hu8E3-28Z 13.7ug、pVSV.G 6.3ug、RRE 16.4ug、REV 16.4ug，加入2200uL DMEM中孵育。对应PEI量为165ul(1ug/ul)，加入2200uL DMEM中孵育5min。将质粒混合液加入PEI孵育液中，混匀，室温孵育20min，得到质粒/PEI混合液。
c)将b)配制的质粒/PEI混合液逐滴加入a)的细胞中，混匀，5h后进行换液，72h后收集病毒上清，以0.45um滤膜过滤后，加入PEG8000，4℃纯化过夜，4000rpm、4℃离心，收集病毒沉淀，得到hu8E3-28Z慢病毒。
3.重组人慢病毒感染T细胞
a)将PBMC培养于AIM-V培基(Gibco,#0870112)+2％human AB serum(Gemini,#100-512)中，加入重组人IL-2(500U/ml)(上海华新生物高技术有限公司)，按照1:1(细胞:磁珠)比例加入CD3/CD28磁珠(Invitrogen，#21013)活化T细胞，48小时后感染。
b)Retronectin(Takara,#T100A)包板4℃过夜，浓度5μg/ml。将活化好的T细胞按照1×10 6个细胞/ml的密度加入Retronectin包好的板中，加入步骤2中收集的高纯度病毒后(MOI≈10)，1800rpm，32℃离心40min，离心完后放入培养箱中培养48h，换液(AIM-Ⅴ培基+2％human AB serum、IL-2 500U/ml)，去磁珠，其后细胞以5×10 5个细胞/ml的密度培养6-10天，得到感染了hu8E3-28Z病毒的T细胞，即表达8E3-28Z(SEQ ID NO:48)的T细胞。
取5×10 5个8E3-28Z T细胞及未感染病毒的T细胞进行流式检测，检测8E3-28Z CAR在T细胞中的的表达，检测仪器为Beckman Coulter Epics XL Flow Cytometer，结果如图11所示，在感染了hu8E3-28Z病毒的T细胞中都能检测到hu8E3-28Z CAR的表达。
实施例9.8E3-28Z的CAR T细胞的细胞毒性检测
取实施例8制备的hu8E3-28Z T细胞，检测对3T3-mFAP及H1080-huFAPα细胞的杀伤，方法如下：
将3T3-mFAP单克隆细胞消化后计数，取表达8E3-28Z CAR的T细胞离心后计数。以3T3-mFAP细胞为靶细胞，效应细胞为表达8E3-28Z CAR的T细胞，效靶比分别为3:1、1:1、1:3，靶细胞数量为10000/孔，根据不同效靶比设定不同数量效应细胞(即30000/孔，10000/孔，3333/孔)。各组均设5个复孔。共孵育18h后通过CytoTox96非放射性细胞毒性试剂盒(Promega，#G1780)检测上清中LDH含量，计算其杀伤活性。具体参照CytoTox96非放射性细胞毒性试剂盒说明书。表达8E3-28Z CAR的T细胞对3T3、H1080及H1080-huFAPα细胞的杀伤毒性实验与3T3-mFAP细胞相同。
如图12所示，表达8E3-28Z CAR的T细胞能够特异性杀伤表达鼠FAPα的3T3-mFAP细胞及表达人FAPα的H1080-huFAPα细胞。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (37)

  1. 一种抗原结合单元，所述抗原结合单元包含轻链CDR区和重链CDR区，所述的重链CDR包含HCDR1，HCDR2，和HCDR3；所述的轻链CDR区包含LCDR1，LCDR2，和LCDR3；
    其中，所述的HCDR1，HCDR2和HCDR3的序列独立选自下组：SEQ ID NO:1-3、7和8，所述的LCDR1，LCDR2和LCDR3的序列独立选自下组：SEQ ID NO:4-6、9和10。
  2. 如权利要求1所述的抗原结合单元，其特征在于，所述的HCDR1具有SEQ ID NO:1或7所示的序列，所述的HCDR2具有SEQ ID NO:2或8所示的序列，所述的HCDR3具有SEQ ID NO:3所示的序列。
  3. 如权利要求2所述的抗原结合单元，其特征在于，所述的HCDR1，HCDR2和HCDR3的序列选自以下任一组：
    A.SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3；
    B.SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:3。
  4. 如权利要求1所述的抗原结合单元，其特征在于，所述的LCDR1具有SEQ ID NO:4或9所示的序列，所述的LCDR2具有SEQ ID NO:5或10所示的序列，所述的LCDR3具有SEQ ID NO:6所示的序列。
  5. 如权利要求4所述的抗原结合单元，其特征在于，所述的LCDR1，LCDR2和LCDR3的序列选自以下任一组：
    A.SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6；
    B.SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:6。
  6. 如权利要求1所述的抗原结合单元，其特征在于，所述的HCDR1具有SEQ ID NO:1或7所示的序列，所述的HCDR2具有SEQ ID NO:2或8所示的序列，所述的HCDR3具有SEQ ID NO:3所示的序列，所述的LCDR1具有SEQ ID NO:4或9所示的序列，所述的LCDR2具有SEQ ID NO:5或10所示的序列，所述的LCDR3具有SEQ ID NO:6所示的序列。
  7. 如权利要求6所述的抗原结合单元，其特征在于，
    所述的HCDR1含有SEQ ID NO:1所示的序列、所述的HCDR2含有SEQ ID NO:2所示的序列、所述的HCDR3含有SEQ ID NO:3所示的序列、所述的LCDR1含有SEQ ID NO:4所示的序列、所述的LCDR2含有SEQ ID NO:5所示的序列、所述的LCDR3含有SEQ ID NO:6所示的序列；或者
    所述的HCDR1含有SEQ ID NO:7所示的序列、所述的HCDR2含有SEQ ID NO:8所示的序列、所述的HCDR3含有SEQ ID NO:3所示的序列、所述的LCDR1含有SEQ ID NO:4所示的序列、所述的LCDR2含有SEQ ID NO:5所示的序列、所述的LCDR3含有SEQ ID NO:6所示的序列；或者
    所述的HCDR1含有SEQ ID NO:1所示的序列、所述的HCDR2含有SEQ ID NO:2所 示的序列、所述的HCDR3含有SEQ ID NO:3所示的序列、所述的LCDR1含有SEQ ID NO:9所示的序列、所述的LCDR2含有SEQ ID NO:10所示的序列、所述的LCDR3含有SEQ ID NO:6所示的序列。
  8. 如权利要求1-7任一所述的抗原结合单元，其特征在于，所述的抗原结合单元具有SEQ ID NO:11或15所示的重链可变区，和SEQ ID NO:13或17所示的轻链可变区。
  9. 权利要求1-8任一所述的抗原结合单元的突变体，与权利要求1-8任一所述的抗原结合单元竞争结合FAPα。
  10. 如权利要求9所述的突变体，其特征在于，所述突变体与权利要求1-8任一所述的抗原结合单元在轻链可变区序列上有90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的相同性；或者，所述突变体与权利要求1-8任一所述的抗原结合单元在在重链可变区序列上有90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的相同性；
    优选地，所述突变体与权利要求1-8任一所述的抗原结合单元在轻链CDR区，例如轻链CDR1或CDR2的序列上有70％以上；优选地，75％以上；更优选地，80％以上的相同性；或者，所述突变体与权利要求1-8任一所述的抗原结合单元在重链CDR区，例如重链CDR1或CDR2的序列上有60％以上；优选地，70％以上；更优选地，80％以上的相同性。
  11. 如权利要求9所述的突变体，其特征在于，所述突变体与权利要求1-8任一所述的抗原结合单元竞争结合FAPα上同一结合表位。
  12. 一种抗原结合单元，所述抗原结合单元包含轻链CDR区和重链CDR区，所述的重链CDR包含HCDR1，HCDR2和HCDR3；所述的轻链CDR区包含LCDR1，LCDR2和LCDR3，其中，所述的HCDR1，HCDR2和HCDR3独立选自与SEQ ID NO:1-3、7、8中任一序列至少80％、优选至少85％、更优选至少90％、更优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％相似性的序列，所述的LCDR1，LCDR2和LCDR3独立选自与SEQ ID NO:4-6、9、10中任一序列至少80％、优选至少85％、更优选至少90％、更优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％相似性的序列。
  13. 如权利要求12所述的抗原结合单元，其特征在于，所述的HCDR1具有SEQ ID NO:1或7所示的序列，所述的HCDR2具有SEQ ID NO:2或8所示的序列，所述的HCDR3具有SEQ ID NO:3所示的序列。
  14. 如权利要求13所述的抗原结合单元，其特征在于，所述的HCDR1，HCDR2和HCDR3的序列选自以下任一组：
    A.SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3；
    B.SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:3。
  15. 如权利要求12所述的抗原结合单元，其特征在于，所述的LCDR1具有SEQ ID NO:4或9所示的序列，所述的LCDR2具有SEQ ID NO:5或10所示的序列，所述的LCDR3具有SEQ ID NO:6所示的序列。
  16. 如权利要求15所述的抗原结合单元，其特征在于，所述的LCDR1，LCDR2和LCDR3的序列选自以下任一组：
    A.SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6；
    B.SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:6。
  17. 如权利要求12所述的抗原结合单元，其特征在于，所述的HCDR1具有SEQ ID NO:1或7所示的序列，所述的HCDR2具有SEQ ID NO:2或8所示的序列，所述的HCDR3具有SEQ ID NO:3所示的序列，所述的LCDR1具有SEQ ID NO:4或9所示的序列，所述的LCDR2具有SEQ ID NO:5或10所示的序列，所述的LCDR3具有SEQ ID NO:6所示的序列。
  18. 如权利要求17所述的抗原结合单元，其特征在于，
    所述的HCDR1含有SEQ ID NO:1所示的序列、所述的HCDR2含有SEQ ID NO:2所示的序列、所述的HCDR3含有SEQ ID NO:3所示的序列、所述的LCDR1含有SEQ ID NO:4所示的序列、所述的LCDR2含有SEQ ID NO:5所示的序列、所述的LCDR3含有SEQ ID NO:6所示的序列；或者
    所述的HCDR1含有SEQ ID NO:7所示的序列、所述的HCDR2含有SEQ ID NO:8所示的序列、所述的HCDR3含有SEQ ID NO:3所示的序列、所述的LCDR1含有SEQ ID NO:4所示的序列、所述的LCDR2含有SEQ ID NO:5所示的序列、所述的LCDR3含有SEQ ID NO:6所示的序列；或者
    所述的HCDR1含有SEQ ID NO:1所示的序列、所述的HCDR2含有SEQ ID NO:2所示的序列、所述的HCDR3含有SEQ ID NO:3所示的序列、所述的LCDR1含有SEQ ID NO:9所示的序列、所述的LCDR2含有SEQ ID NO:10所示的序列、所述的LCDR3含有SEQ ID NO:6所示的序列。
  19. 一种抗原结合单元，其特征在于，所述的抗原结合单元具有与SEQ ID NO:11或15所示的重链可变区的序列具有至少80％、优选至少85％、更优选至少90％、更优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％相似性的序列，和与SEQ ID NO:13或17所示的轻链可变区具有至少80％、优选至少85％、更优选至少90％、更优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％相似性的序列。
  20. 如权利要求1-19任一项所述的抗原结合单元或抗原结合单元的突变体，其特征在于，所述的抗原结合单元是单克隆抗体、全人抗体、人源化抗体、嵌合抗体。
  21. 如权利要求1-19所述的抗原结合单元，其特征在于，所述的抗原结合单元是scFv、Fv、Fab、(Fab) 2或者单域抗体。
  22. 编码权利要求1-21中任一所述的抗原结合单元的核酸。
  23. 一种表达载体，其包含权利要求22所述的核酸。
  24. 一种宿主细胞，其包含权利要求23所述的表达载体或基因组中整合有权利要求22所述的核酸。
  25. 一种双价蛋白，由权利要求21所述的scFv序列与一个或多个重链恒定区域融合所形成具有人免疫球蛋白Fc区的抗体；
    优选地，所述的人免疫球蛋白Fc区连接有缀合物；优选的，所述缀合物优选自荧光染 料、细胞毒素、放射性同位素。
  26. 一种多功能免疫辍合物，其特征在于，所述的多功能免疫辍合物包括:
    权利要求1-21任一所述的抗原结合单元，以及与之连接的功能性分子；其中，所述的功能性分子选自:
    (a)靶向肿瘤表面标志物的分子，包括结合免疫细胞表面标志物的抗体或配体；较佳地，所述的免疫细胞表面标志物包括:CD3，CD16，CD28；更佳地，所述的结合免疫细胞表面标志物的抗体是抗CD3抗体；最佳地，所述的靶向免疫细胞的表面标志物的分子是结合T细胞表面标志物的抗体；
    (b)抑制肿瘤的分子,包括抗肿瘤的细胞因子或抗肿瘤的毒素，较佳的，所述的细胞因子包括:IL-12、IL-15、I型干扰素、TNF-alpha；
    (c)靶向免疫细胞的表面标志物的分子或可检测标记物。
  27. 一种嵌合抗原受体，其特征在于，所述嵌合抗原受体的胞外域包含权利要求1-21任一所述的抗原结合单元，该抗原结合单元优选单链抗体或单域抗体。
  28. 如权利要求27所述的嵌合抗原受体，其特征在于，所述的嵌合抗原受体包括如下顺序连接的抗体，跨膜区和胞内信号区:
    权利要求1-21任一所述的抗原结合单元、CD8和CD3ζ；
    权利要求1-21任一所述的抗原结合单元、CD8、CD137和CD3ζ；
    权利要求1-21任一所述的抗原结合单元、CD28分子的跨膜区、CD28分子的胞内信号区和CD3ζ；或
    权利要求1-21任一所述的抗原结合单元、CD28分子的跨膜区、CD28分子的胞内信号区、CD137和CD3ζ。
  29. 编码权利要求27或28所述的嵌合抗原受体的核苷酸序列。
  30. 一种表达载体，其特征在于，包含权利要求29所述的核苷酸序列。
  31. 一种病毒，其特征在于，所述的病毒包含权利要求30所述的表达载体。
  32. 一种基因修饰的免疫细胞，其特征在于，
    其转导有权利要求29所述的核苷酸序列、
    或权利要求30所述的表达载体、
    或权利要求31所述的病毒、
    或其表达权利要求27或28所述的嵌合抗原受体。
  33. 如权利要求32所述的基因修饰的免疫细胞，其特征在于，还表达有除嵌合抗原受体之外的其他序列，所述其他序列包括细胞因子、另一种嵌合抗原受体、趋化因子受体、降低PD-1表达的siRNA或者阻断PD-L1的蛋白、TCR、或安全开关；
    较佳地，所述的细胞因子包括IL-12、IL-15、IL-21、或I型干扰素；
    较佳地，所述趋化因子受体包括CCR2、CCR5、CXCR2、或CXCR4；
    较佳地，所述安全开关包括iCaspase-9、Truancated EGFR或RQR8。
  34. 如权利要求1-21任一所述的抗原结合单元、或权利要求25所述的双价蛋白、或权 利要求26所述的免疫辍合物、或权利要求27或28所述的嵌合抗原受体、或权利要求29所述的核苷酸序列、或权利要求30所述的表达载体、或权利要求31所述的病毒、或权利要求32或33所述的免疫细胞的用途，用于制备治疗、预防或诊断高表达FAPα相关的疾病的药物或试剂。
  35. 如权利要求34所述的用途，所述的高表达FAPα相关的疾病是肿瘤。
  36. 如权利要求35所述的用途，所述的肿瘤是乳腺癌、卵巢癌、肺癌、结肠癌、胰腺癌、皮肤黑色素瘤、肾癌、膀胱癌。
  37. 一种药物组合物，其特征在于，所述药物组合物包含权利要求1-21中任一所述的抗原结合单元或编码该抗原结合单元的核酸；或
    权利要求25中所述的双价蛋白；或
    权利要求26中所述的免疫辍合物或编码该辍合物的核酸；或
    权利要求27或28所述的嵌合抗原受体或编码该嵌合抗原受体的核酸；或
    权利要求32或33中所述的基因修饰的免疫细胞。
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US11872291B2 (en) 2016-12-14 2024-01-16 Purdue Research Foundation Fibroblast activation protein (FAP)-targeted imaging and therapy
EP4034554A4 (en) * 2019-09-23 2023-10-25 The Trustees of the University of Pennsylvania DESTRUCTION OF TUMOR TISSUE BY TARGETING FIBROBLAST ACTIVATION PROTEIN (FAP)
JP2023533042A (ja) * 2020-08-11 2023-08-01 カナプ セラピューティクス インコーポレイテッド Ｉｌ－１２及び抗ｆａｐ抗体を含む融合タンパク質、並びにその使用
WO2022214089A1 (zh) 2021-04-08 2022-10-13 克莱格医学有限公司 细胞免疫治疗的应用
WO2023125796A1 (en) * 2021-12-30 2023-07-06 Concept To Medicine Biotech Co., Ltd. Human antibodies against fap-alpha

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