CN117043184A - Nanometer antibody for resisting Human Serum Albumin (HSA) and application thereof - Google Patents

Nanometer antibody for resisting Human Serum Albumin (HSA) and application thereof Download PDF

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CN117043184A
CN117043184A CN202280022765.8A CN202280022765A CN117043184A CN 117043184 A CN117043184 A CN 117043184A CN 202280022765 A CN202280022765 A CN 202280022765A CN 117043184 A CN117043184 A CN 117043184A
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antigen
antibody
cancer
binding fragment
antibodies
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成广存
葛虎
曹卓晓
唐任宏
任晋生
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Nanjing Zaiming Pharmaceutical Co ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans

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Abstract

The invention discloses a nano antibody specifically combined with Human Serum Albumin (HSA) and application thereof, and particularly discloses an alpaca capable of combining with Human Serum Albumin (HSA) and a humanized nano antibody or an antigen binding fragment thereof, and has better affinity with human, mouse and monkey serum albumin, and has important significance for treating cancers and autoimmune diseases.

Description

Nanometer antibody for resisting Human Serum Albumin (HSA) and application thereof Technical Field
The invention relates to the field of antibodies, in particular to an HSA alpaca nano antibody and a humanized antibody thereof.
Background
Pharmacokinetics is an important parameter in the drug development process. For macromolecular drugs, a longer serum half-life means lower dosing frequency and dosage, and the bioavailability of the drug is correspondingly improved, thereby producing better therapeutic effects. Currently, methods for extending the half-life of drugs are Fc fusion, polyethylene glycol PEG coupling, and Human Serum Albumin (HSA) fusion.
HSA is a novel molecule with a molecular weight of 67kD, constituting two-thirds of the mass of serum proteins, and having many physiological properties. Serum albumin carries many endogenous substances such as inorganic ions, fatty acids, bilirubin, vitamins, hormones and steroids. And is also a carrier for many exogenous drugs. The HSA protein has 17 disulfide bonds inside, so that the whole protein has good stability, the HSA has the advantages of prolonging half-life period, promoting the drug to permeate blood brain barrier and the like, is not easy to permeate kidney glomeruli, has half-life period of 2 weeks in plasma, is widely distributed in vivo and has no immunogenicity, and is a relatively ideal protein drug carrier. However, the HSA fusion protein has limited application range to some extent due to its large molecular weight, low expression level, difficulty in purification, and the like.
Single domain antibodies are a class of antibodies that naturally lack the light chain but only the heavy chain variable region, and have a relative molecular weight of only 15kD, which is only about 1/10 of the molecular weight of a conventional antibody, and thus are also referred to as nanobodies or VHH antibodies. Nanobodies are the smallest unit of antigen binding with complete function that is currently available. Because of its small molecular weight, good stability and flexible chemical properties, it is often used as an ideal form for building bispecific antibodies.
Developing a nano antibody targeting HSA, and constructing a fusion protein by taking the nano antibody as a structural block for prolonging the half life of an active drug, becomes an important drug development strategy, such as a protein drug Vobarilizumab targeting IL6R, wherein the anti-HSA nano antibody is contained as the structural block.
The invention relates to a humanized HSA nano antibody with a brand new sequence, which can specifically identify serum albumin of human, monkey and mouse and has good binding activity.
Disclosure of Invention
The present invention discloses HSA nanobody or antigen binding fragment, multispecific antigen binding molecule, chimeric antigen receptor, immune effector cell, nucleic acid fragment, vector, cell, composition, preparation method, pharmaceutical use, and disease treatment method.
In some embodiments, an isolated nanobody or antigen-binding fragment that specifically binds Human Serum Albumin (HSA) comprises a combination of CDRs of the heavy chain variable region comprising: HCDR1, HCDR2 and HCDR3; the HCDR1, HCDR2 and HCDR3 have any sequence combination selected from the group consisting of:
each of HCDR1, HCDR2 and HCDR3 is encoded according to a current analytical method of KABAT, chothia or IMGT; preferably, the substitution is a conservative amino acid substitution.
In particular, for example, nanobodies or antigen-binding fragments of the invention, wherein:
(1) The HCDR1, HCDR2 and HCDR3 are respectively shown as SEQ ID NO.31, 32 and 33;
(2) The HCDR1, HCDR2 and HCDR3 are respectively shown as SEQ ID NO.34, 35 and 36;
(3) The HCDR1, HCDR2 and HCDR3 are respectively shown as SEQ ID NO.37, 38 and 36;
(4) The HCDR1, HCDR2 and HCDR3 are respectively shown as SEQ ID NO.39, 40 and 41;
(5) The HCDR1, HCDR2 and HCDR3 are respectively shown as sequences shown in SEQ ID NO.42, 43 and 44;
(6) The HCDR1, HCDR2 and HCDR3 are respectively shown as sequences shown in SEQ ID NO.45, 46 and 44;
(7) The HCDR1, HCDR2 and HCDR3 are respectively shown as SEQ ID NO.47, 48 and 49;
(8) The HCDR1, HCDR2 and HCDR3 are respectively shown as SEQ ID NO.50, 51 and 52;
(9) The HCDR1, HCDR2 and HCDR3 are respectively shown as SEQ ID NO.53, 54 and 52; or alternatively, the first and second heat exchangers may be,
(10) The HCDR1, HCDR2 and HCDR3 are a sequence combination having 1, 2, 3 or more amino acid insertions, deletions and/or substitutions compared to a sequence combination of (1) - (9) above; substitutions are preferred, more preferably conservative amino acid residue substitutions.
In another specific embodiment, the framework regions of the heavy chain variable regions of the nanobodies or antigen-binding fragments of the invention are derived from human germline heavy chains, wherein:
(1) The framework region sequence is derived from the combined sequence of human germline heavy chains IGHV3-23 x 04 and IGHJ2 x 01; which comprises SEQ ID NO:28, the FR1, FR2, FR3 regions of igfv 3-23 x 04 and SEQ ID NO:29 to igfj 2 x 01;
(2) The framework region sequence is derived from the combined sequence of human germline heavy chains IGHV3-23 x 04 and IGHJ3 x 01; which comprises SEQ ID NO:28, the FR1, FR2, FR3 regions of igfv 3-23 x 04 and SEQ ID NO:30, the FR4 region of IGHJ3 x 01.
In another specific embodiment, the framework regions of the heavy chain variable region of the nanobody or antigen-binding fragment of the invention further comprise one or more mutations according to Kabat numbering selected from the group consisting of:
(1) E1Q, S30N, V F, I70V, S3756A, Q82E, S85N, A97R, K98Q, W109R or L114Q; preferably including S30N, V37F, I70V, A97R, K98Q and W109R; or preferably S30N, V37F, I70V, A97R, K98Q, W109R and E1Q; or preferably comprises S30N, V37F, I70V, A97R, K98Q, W109R and S75A; or preferably S30N, V37F, I70V, A97R, K98Q, W109R and Q82E; or preferably comprises S30N, V37F, I70V, A97R, K98Q, W109R and S85N; or preferably comprises S30N, V37F, I70V, A97R, K98Q, W109R and L114Q;
(2) E1Q, A23V, F V, T28D, S R, S6275A, R87K, A88F, T M, A97V, K98Q, W P or M116Q; preferably including E1Q, F27V, T D, S R, S75A, T91M, A97V, K98Q, W P and M116Q; preferably including F27V, T28D, S30R, S A, T91M, A97V, K3598Q, W111P and M116Q; preferably including F27V, T28D, S R, T91M, A97V, K98Q, W P and M116Q; preferably including F27V, T28D, S30R, R87K, A88F, T91M, A97V, K98Q, W P and M116Q; preferably including E1Q, A23V, F27V, T D, S30R, S A, A97V, K Q and W111P; preferably comprising a23V, F27V, T28D, S R, S30 6275A, A97V, K Q and W111P; preferably including F27V, T28D, S30R, R87K, A F, T91M, A97V, K Q and W111P;
(3) V2L, T28A, S75A, R87K, A88P, A97S, K I or W104S; preferably comprising a97S and K98I; preferably comprising a97S, K I and W104S; preferably comprising V2L, A97S, K I and W104S; preferably including T28A, A97S, K I and W104S; preferably comprising V2L, T28A, A97S, K98I and W104S; preferably, R87K, A88P, A97S, K98I and W104S; preferably comprising V2L, T28A, S A, A97S, K98I and W104S; preferably V2L, T28A, R87K, A88P, A97S, K98I and W104S.
In particular, the nanobody or antigen-binding fragment of the invention further comprises:
(1) The variable region has a sequence shown in SEQ ID NO 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27;
(2) An amino acid sequence having at least 90% identity, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity, to the sequence shown in (1) above; or alternatively, the first and second heat exchangers may be,
(3) The framework regions of the nanobody or antigen-binding fragment have at least 90% identity, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity, to the framework regions of the amino acid sequences shown in SEQ ID NOs 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27.
In a preferred embodiment, the nanobody or antigen-binding fragment of the invention binds to human serum albumin, monkey serum albumin and/or rat serum albumin; alternatively, the antibody or antigen binding fragment binds to human serum albumin, monkey serum albumin, and/or rat serum albumin with a dissociation constant (KD) of less than 1.00E-8M, 2.00E-8M, 3.00E-8M, 1.00E-9M, 2.00E-09M, 3.00E-9M, 4.00E-09M, 5.00E-09M, 6.00E-09M, 7.00E-09M, 8.00E-09M, 9.00E-09M, 1.00E-10M, 2.00E-10M, 3.00E-10M, 4.00E-10M, 5.00E-10M, 6.00E-10M, 7.00E-10M, 8.00E-10M, 9.00E-10M, 1.00E-11M, 2.00E-11M, 3.00E-11M, 4.00E-11M, 1.00E-10M, 2.00E-11M, 3.00E-12.00E-12M, 12.00E-12M, 3.00E-12.00E-12M;
alternatively, the nanobody or antigen-binding fragment binds to mouse serum albumin or does not bind.
In a preferred embodiment, the antibody or antigen binding fragment of the invention comprises the sequence of the constant region of any one of antibodies IgG1, igG2, igG3, igG4, igA, igM, igE or IgD; preferably comprising the sequences of the constant regions of antibodies IgG1, igG2, igG3 or IgG 4.
In a preferred embodiment, the antibody or antigen binding fragment of the invention further comprises an antibody constant region sequence in the absence of a CH1 fragment.
In a preferred embodiment, the antibody or antigen-binding fragment of the invention further comprises an antibody constant region sequence having CH2 and CH3 fragments, or the antibody or antigen-binding fragment further comprises an antibody Fc region;
the antibody constant region or antibody Fc region is linked to the antibody or antigen binding fragment with or without a linking peptide;
alternatively, the antibody constant region or antibody Fc region is from a camelid, mouse, rat, rabbit, sheep or human;
alternatively, the antibody constant region or antibody Fc region is from IgG, igA, igM, igD or IgE.
In a preferred embodiment, the antibody or antigen binding fragment thereof of the invention is chimeric or humanized or fully human; preferably, the antibody or antigen binding fragment is selected from the group consisting of monoclonal antibodies, polyclonal antibodies, natural antibodies, engineered antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), monovalent antibodies, multivalent antibodies, full length antibodies, antibody fragments, naked antibodies, conjugated antibodies, humanized antibodies, fully human antibodies, fab ', F (ab') 2, fd, fv, scFv, diabodies (diabodies), or single domain antibodies.
In a preferred embodiment, the antibody or antigen binding fragment thereof of the invention is further conjugated to a therapeutic agent or tracer; preferably, the therapeutic agent is selected from the group consisting of a radioisotope, a chemotherapeutic agent or an immunomodulator, and the tracer is selected from the group consisting of a radiocontrast agent, a paramagnetic ion, a metal, a fluorescent label, a chemiluminescent label, an ultrasound contrast agent or a photosensitizer.
In a preferred embodiment, the invention also provides a multispecific antigen-binding molecule; preferably, the multispecific antigen-binding molecule comprises a first antigen-binding moiety comprising an antibody or antigen-binding fragment of any one of the above, and a second antigen-binding moiety that specifically binds to an antigen other than HSA or to an HSA epitope different from the first antigen-binding moiety;
preferably, the additional antigen is selected from the group consisting of CD2, CD3, CD4, CD5, CD7, CD8, CD11a, CD11b, CD18, CD19, CD20, CD23, CD25, CD33, CD38, CD40, CD45, CDW52, CD69, CD70, CD134 (OX 40), ICOS, BCMP7, CD137, CD27L, CDCP1, DPCR1, dudulin2, FLJ20584, FLJ40787, HEK2, KIAA0634, KIAA0659, KIAA1246, KIAA1455, LTBP2, LTK, MAL2, MRP2, MSLN;
Preferably, the multispecific antibody is a bispecific antibody, a trispecific antibody, or a tetraspecific antibody.
In a preferred embodiment, the invention provides a Chimeric Antigen Receptor (CAR); preferably, the chimeric antigen receptor comprises at least an extracellular antigen binding domain comprising the HSA antibody or antigen binding fragment of any of the above, a transmembrane domain, and an intracellular signaling domain.
In a preferred embodiment, the invention provides an immune effector cell; preferably, the immune effector cell comprises the chimeric antigen receptor described above or a nucleic acid fragment comprising the chimeric antigen receptor described above;
preferably, the immune effector cell is selected from T cells, NK cells (natural killer cell), NKT cells (natural killer cell), monocytes, macrophages, dendritic cells or mast cells; the T cells may be selected from inflammatory T cells, cytotoxic T cells, regulatory T cells (tregs) or helper T cells;
preferably, the immune effector cell is an allogeneic immune effector cell or an autoimmune cell.
In a preferred embodiment, the present invention provides an isolated nucleic acid molecule encoding a nanobody, an antigen-binding fragment, or any combination thereof, as described in any of the above, a multispecific antigen-binding molecule, or a chimeric antigen receptor, as described above.
In some embodiments, the invention provides an expression vector comprising the isolated nucleic acid molecule of the invention described above.
In some embodiments, the invention provides a host cell comprising an isolated nucleic acid molecule or expression vector of the invention described above.
In a preferred embodiment, the host cell is a eukaryotic cell or a prokaryotic cell; more preferably, the host cell is derived from mammalian cells, yeast cells, insect cells, E.coli and/or B.subtilis; more preferably, the host cell is selected from the group consisting of an Expi293F cell.
In some embodiments, the invention provides a method of producing an antibody or antigen-binding fragment or multispecific antigen-binding molecule, culturing a host cell of the invention described above under appropriate conditions, and isolating the antibody or antigen-binding fragment or multispecific antigen-binding molecule.
In some embodiments, the invention provides a method of preparing an immune effector cell, introducing a nucleic acid fragment of the CAR described above into the immune effector cell, preferably the method further comprises initiating expression of the CAR described above by the immune effector cell.
In some embodiments, the invention provides a pharmaceutical composition comprising an antibody or antigen binding fragment of the invention described above, a multispecific antigen-binding molecule of the invention described above, a chimeric antigen receptor of the invention described above, an immune effector cell of the invention described above, an isolated nucleic acid molecule of the invention described above, an expression vector of the invention described above, a cell of the invention described above, or a product (e.g., an antibody and antigen binding fragment) made by the method of the invention described above, and a pharmaceutically acceptable carrier.
In a preferred embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent or adjuvant; more preferably, the pharmaceutical composition further comprises an additional anti-tumor agent.
In some embodiments, the invention provides the use of an antibody or antigen binding fragment as described above, a multispecific antigen binding molecule as described above, a chimeric antigen receptor as described above, an immune effector cell as described above, an isolated nucleic acid molecule as described above, an expression vector as described above, a cell as described above, a product (e.g., an antibody and antigen binding fragment) as described above, or a pharmaceutical composition as described above, in the manufacture of a medicament for the prevention and/or treatment of a neoplastic disease or inflammatory disease, preferably melanoma, clear cell sarcoma, head and neck cancer, bladder cancer, breast cancer, colon cancer, ovarian cancer, endometrial cancer, gastric cancer, pancreatic cancer, kidney cancer, prostate cancer, salivary gland cancer, lung cancer, liver cancer, skin cancer, or brain cancer; the inflammatory disease is preferably multiple sclerosis, psoriasis, myasthenia gravis, uveitis, systemic lupus erythematosus, or rheumatoid arthritis.
In some embodiments, the invention provides a method of preventing and/or treating a neoplastic disease or inflammatory disease comprising administering to a patient in need thereof an antibody or antigen binding fragment of the invention described above, a multispecific antigen binding molecule of the invention described above, a chimeric antigen receptor of the invention described above, an immune effector cell of the invention described above, an isolated nucleic acid molecule of the invention described above, an expression vector of the invention described above, a cell of the invention described above, a product made by a method of the invention described above, or a pharmaceutical composition of the invention described above; the neoplastic disease is preferably melanoma, clear cell sarcoma, head and neck cancer, bladder cancer, breast cancer, colon cancer, ovarian cancer, endometrial cancer, gastric cancer, pancreatic cancer, renal cancer, prostate cancer, salivary gland cancer, lung cancer, liver cancer, skin cancer, or brain cancer; the inflammatory disease is preferably multiple sclerosis, psoriasis, myasthenia gravis, uveitis, systemic lupus erythematosus, or rheumatoid arthritis.
In some embodiments, the invention provides an antibody or antigen binding fragment as described above, a multispecific antigen binding molecule as described above, a chimeric antigen receptor as described above, an immune effector cell as described above, an isolated nucleic acid molecule as described above, an expression vector as described above, a cell as described above, a product (e.g., an antibody and antigen binding fragment) as described above, or a pharmaceutical composition as described above for use in preventing and/or treating a neoplastic disease or inflammatory disease; the neoplastic disease is preferably melanoma, clear cell sarcoma, head and neck cancer, bladder cancer, breast cancer, colon cancer, ovarian cancer, endometrial cancer, gastric cancer, pancreatic cancer, renal cancer, prostate cancer, salivary gland cancer, lung cancer, liver cancer, skin cancer, or brain cancer; the inflammatory disease is preferably multiple sclerosis, psoriasis, myasthenia gravis, uveitis, systemic lupus erythematosus, or rheumatoid arthritis.
In some embodiments, the invention provides a kit comprising an antibody or antigen binding fragment of the invention described above, a multispecific antigen-binding molecule of the invention described above, a chimeric antigen receptor of the invention described above, an immune effector cell of the invention described above, an isolated nucleic acid molecule of the invention described above, an expression vector of the invention described above, a cell of the invention described above, or a product (e.g., an antibody and antigen binding fragment) made by a method of the invention described above, or a pharmaceutical composition of the invention described above, and instructions for use.
Definition and description of terms
Unless otherwise indicated, terms used herein have meanings commonly understood by one of ordinary skill in the art. For a term explicitly defined herein, the meaning of that term controls the definition.
As used herein, the term "antibody" (Ab) refers to immunoglobulin molecules that specifically bind to or are immunoreactive with an antigen of interest, including polyclonal, monoclonal, genetically engineered and other modified forms of the antibody (including but not limited to chimeric antibodies, humanized antibodies, fully human antibodies, heteroconjugate antibodies (e.g., bispecific, trispecific and tetraspecific antibodies, diabodies, trisomy and tetrasomy), antibody conjugates, and antigen-binding fragments of the antibody (including, e.g., fab ', F (Ab ') 2, fab, fv, rIgG and scFv fragments). Furthermore, unless otherwise indicated, the term "monoclonal antibody" (mAb) is intended to include intact antibody molecules capable of specifically binding to the target protein as well as incomplete antibody fragments (e.g., fab and F (Ab ') 2 fragments that lack the Fc fragment of the intact antibody (that is cleared more rapidly from the animal cycle), and thus lack Fc-mediated effector functions (effector function) (see Wahl et al, j. Nucl. Med.24:316,1983; the disclosure of which is incorporated herein).
The "antibody" herein may be derived from any animal, including but not limited to humans and non-human animals, which may be selected from primates, mammals, rodents and vertebrates, such as camelids, llamas, primo-ostris, alpacas, sheep, rabbits, mice, rats or chondrilleids (e.g. shark).
The term "monospecific" herein refers to having one or more binding sites, wherein each binding site binds to the same epitope of the same antigen.
The term "multispecific" herein refers to having at least two antigen-binding sites, each of which binds to a different epitope of the same antigen or to a different epitope of a different antigen. Thus, terms such as "bispecific," "trispecific," "tetraspecific," and the like refer to the number of different epitopes to which an antibody/antigen binding molecule can bind.
"full length antibody", "intact antibody" and "whole antibody" are used interchangeably herein to mean that they have a structure substantially similar to the structure of a native antibody.
As used herein, the term "antigen binding fragment" refers to one or more antibody fragments that retain the ability to specifically bind a target antigen. The antigen binding function of an antibody may be performed by a fragment of a full-length antibody. The antibody fragment may be a Fab, F (ab') 2, scFv, SMIP, diabody, triabody, affibody (affibody), nanobody, aptamer, or domain antibody. Examples of binding fragments that encompass the term "antigen-binding fragment" of an antibody include, but are not limited to: (i) Fab fragment, a monovalent fragment consisting of VL, VH, CL and CH1 domains; (ii) A F (ab) 2 fragment, a bivalent fragment comprising two Fab fragments linked at a hinge region by a disulfide bond; (iii) an Fd fragment consisting of VH and CH1 domains; (iv) Fv fragments consisting of the VL and VH domains of the antibody single arm; (V) a dAb comprising VH and VL domains; (vi) dAb fragments consisting of VH domains (Ward et al Nature 341:544-546,1989); (vii) a dAb consisting of a VH or VL domain; (viii) an isolated Complementarity Determining Region (CDR); and (ix) a combination of two or more isolated CDRs, which may optionally be connected by a synthetic linker. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, these two domains can be joined, using recombinant methods, by a linker that enables them to be made into a single protein chain in which the VL and VH regions pair to form a monovalent molecule (known as a single chain Fv (scFv); see, e.g., bird et al, science 242:423-426,1988, and Huston et al, proc. Natl. Acad. Sci. USA 85:5879-5883,1988). These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and these fragments are screened for use in the same manner as whole antibodies. Antigen binding fragments may be produced by recombinant DNA techniques, enzymatic or chemical cleavage of intact immunoglobulins, or in some embodiments by chemical peptide synthesis procedures known in the art.
As used herein, the term "serum albumin" refers to the most abundant blood protein and functions as a carrier protein for steroids, fatty acids and thyroid hormones in the blood and plays a major role in stabilizing extracellular fluid volume. For purposes of nomenclature only and not limitation, an exemplary sequence of human serum albumin is shown in NCBI GenBank accession No. AEE 60908. It is understood that references to "serum albumin" or "albumin" include preproalbumin (preproalbumin) which comprises an N-terminal peptide, a proprotein (proalbumin) and a secreted albumin. Albumin comprises three homologous domains, each of which is the product of two subdomains having a common structural motif. Domains I, II and III can be defined with reference to human serum albumin. For example, domain I comprises amino acids 1 (±1 to 15 amino acids) to 194 (±1 to 15 amino acids) of human serum albumin, domain II comprises amino acids 192 (±1 to 15 amino acids) to 387 (±1 to 15 amino acids) of human serum albumin, and domain III comprises amino acid residues 381 (±1 to 15 amino acids) to 585 (±1 to 15 amino acids) of human serum albumin. The phrase "±1 to 15 amino acids" means that the amino acid residue may deviate from the C-terminus and/or N-terminus of the amino acid position referred to by 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 amino acids. Exemplary domains I, II and III are described by Dockal et al (The Journal of Biological Chemistry,1999, volume 274 (41): 29303-29310) and Kjeldsen et al (Protein Expression andPurification,1998, volume 13: 163-169).
As used herein, the term "bispecific antibody" refers to an antibody, typically a human or humanized antibody, having monoclonal binding specificity for at least two different antigens. In the present invention, one of the binding specificities may be detected against an epitope of HSA and the other may be detected against another epitope of HSA or against any other antigen than HSA, e.g. against a cell surface protein, a receptor subunit, a tissue specific antigen, a virus-derived protein, a virus-encoded envelope protein, a bacteria-derived protein or a bacteria-surface protein, etc.
As used herein, the term "chimeric" antibody refers to an antibody having a variable sequence derived from an immunoglobulin of one origin organism (e.g., rat or mouse) and constant regions derived from an immunoglobulin of a different organism (e.g., human). Methods for producing chimeric antibodies are known in the art. See, e.g., morrison,1985, science 229 (4719): 1202-7; oi et al, 1986,Bio Techniques 4:214-221; gilles et al 1985J Immunol Methods 125:191-202; the above is incorporated by reference herein.
As used herein, the term "nanobody" refers to a natural heavy chain antibody lacking a light chain, whose variable region is cloned to give a single domain antibody consisting of only the heavy chain variable region, also known as VHH (Variable domain of heavy chain of heavy chain antibody), which is the smallest functional antigen-binding fragment. For further description of VHH and nanobodies, reference is made to the review article by Muyldermans (2001,Reviews in Molecular Biotechnology 74:277-302), and to the following patent applications mentioned as general background: WO 94/04678, WO 95/04079 and WO 96/34103 at the university of Brussell freedom; WO 94/25591, WO 99/37681, WO 00/40968, WO 00/43507, WO 00/65057, WO 01/40310, WO 01/44301, EP 1134231 and WO 02/48193 in the form of Co-pending U.S. Pat. No.; WO 97/49505, WO 01/21817, WO 03/035694, WO 03/054016 and WO 03/055527 of Vlaams Instituut voor Biotechnologie (VIB); WO03/050531 to Alganomics N.V. and Ablynx N.V.; WO 01/90190 to Canadian national research council; WO 03/025020 (=ep 1433793) of Institute of Antibodies; and Ablynx N.V. WO 04/041687, WO 04/041682, WO 04/041685, WO 04/041683, WO 04/062551, WO 05/044858, WO 06/40153, WO 06/079372, WO 06/122786, WO 06/122787 and WO 06/122825, and Ablynx N.V. further published patent applications. Reference is also made to the further prior art mentioned in these applications, in particular to the list of references mentioned on pages 41-43 of international application WO 06/040153, which list and references are incorporated herein by reference. Nanobodies (particularly VHH sequences and partially humanized nanobodies) can be characterized, inter alia, by the presence of one or more "feature residues" in one or more framework sequences, as described in these references. Further descriptions of nanobodies, including humanization and/or camelization of nanobodies, as well as other modifications, parts or fragments, derivatives or "nanobody fusions", multivalent constructs (including some non-limiting examples of linker sequences) and different modifications that increase half-life of nanobodies and their formulations, can be found, for example, in WO 08/101985 and WO 08/142164. For further general description of nanobodies, reference is made to the prior art cited herein, for example as described in WO08/020079 (page 16).
As used herein, the term "complementarity determining region" (CDR) refers to a hypervariable region found in both the light and heavy chain variable domains. The more conserved portions of the variable domains are called the Framework Regions (FR). As understood in the art, the amino acid positions representing the hypervariable regions of an antibody may vary depending on the context and various definitions known in the art. Some positions within the variable domain may be considered heterozygous hypervariable positions, as these positions may be considered to be within a hypervariable region under one set of criteria (e.g. IMGT or KABAT) and outside a hypervariable region under a different set of criteria (e.g. KABAT or IMGT). One or more of these locations may also be found in the extended hypervariable region. The invention includes antibodies comprising modifications in the positions of these heterozygous hypermutations. The variable domains of the natural heavy and light chains each comprise four framework regions, principally in a lamellar configuration, which are linked by three CDRs (CDR 1, CDR2 and CDR 3) that form loops connecting the lamellar structure and in some cases form part of the lamellar structure. The CDRs in each chain are held closely together by the FR regions in sequence FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and with CDRs from other antibody chains contribute to the formation of the antigen binding site of the antibody (see Kabat et al Sequences of Protein sofImmunological Interest, national Institute of Health, bethesda, md.1987; incorporated herein by reference).
The term "Kabat numbering system" herein generally refers to the immunoglobulin alignment and numbering system proposed by Elvin a.kabat (see, e.g., kabat et al, sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md, 1991).
The term "Chothia numbering system" herein generally refers to the immunoglobulin numbering system proposed by Chothia et al, which is a classical rule for identifying the boundaries of CDR regions based on the position of structural loop regions (see, e.g., chothia & Lesk (1987) J.mol. Biol.196:901-917; chothia et al (1989) Nature 342:878-883).
The term "IMGT numbering system" herein generally refers to the immunoglobulin numbering system proposed by Chothia et al, which is a classical rule for identifying the boundaries of CDR regions based on the position of structural loop regions (see, e.g., chothia & Lesk (1987) J.mol. Biol.196:901-917; chothia et al (1989) Nature 342:878-883).
As used herein, the term "monoclonal antibody" refers to an antibody derived from a single clone (including any eukaryotic, prokaryotic, or phage clone), and is not limited to the method of production of the antibody.
As used herein, the term "VH" refers to the variable region of an immunoglobulin heavy chain of an antibody (including the heavy chain of Fv, scFv, or Fab). The term "VL" refers to the variable region of an immunoglobulin light chain (including the light chain of Fv, scFv, dsFv or Fab).
The term "heavy chain constant region" herein refers to the carboxy-terminal portion of an antibody heavy chain that does not directly participate in binding of the antibody to an antigen, but exhibits effector functions, such as interactions with Fc receptors, that have more conserved amino acid sequences relative to the variable domains of the antibody. The "heavy chain constant region" comprises at least one of: a CH1 domain, a hinge region, a CH2 domain, a CH3 domain, or a variant or fragment thereof. "heavy chain constant regions" include "full length heavy chain constant regions" having a structure substantially similar to that of a natural antibody constant region and "heavy chain constant region fragments" including only a portion of the "full length heavy chain constant region. Illustratively, a typical "full length antibody heavy chain constant region" consists of a CH1 domain-hinge region-CH 2 domain-CH 3 domain; when the antibody is IgE, it further comprises a CH4 domain; when an antibody is a heavy chain antibody, then it does not include a CH1 domain. Exemplary, a typical "heavy chain constant region fragment" may be selected from a CH1, fc, or CH3 domain.
The term "light chain constant region" herein refers to the carboxy-terminal portion of an antibody light chain, which is not directly involved in binding of an antibody to an antigen, and which may be selected from a constant kappa domain or a constant lambda domain.
The term "Fc" herein refers to the carboxy-terminal portion of an antibody that is formed by the proteolytic hydrolysis of papain in to an intact antibody, typically comprising the CH3 and CH2 domains of the antibody. The Fc region includes, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary slightly, the Fc region of a human IgG heavy chain is generally defined as extending from amino acid residue position Cys226 or from Pro230 to its carboxy terminus. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during production or purification of the antibody, or by recombinant engineering of the nucleic acid encoding the heavy chain of the antibody, and thus the Fc region may or may not include Lys447.
The term "humanized antibody" as used herein refers to a genetically engineered non-human antibody whose amino acid sequence is modified to increase homology with the sequence of a human antibody. Typically, all or part of the CDR regions of a humanized antibody are derived from a non-human antibody (donor antibody) and all or part of the non-CDR regions (e.g., variable region FR and/or constant regions) are derived from a human immunoglobulin (acceptor antibody). Humanized antibodies generally retain or partially retain the desired properties of the donor antibody, including, but not limited to, antigen specificity, affinity, reactivity, ability to enhance immune cell activity, ability to enhance immune responses, and the like.
The term "fully human antibody" herein refers to an antibody having variable regions in which both the FR and CDR are derived from human germline immunoglobulin sequences. Furthermore, if the antibody comprises constant regions, the constant regions are also derived from human germline immunoglobulin sequences. Fully human antibodies herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, herein "fully human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been grafted onto human framework sequences.
The term "naked antibody" herein refers to an antibody that is not linked, fused or conjugated to another agent or molecule (e.g., a label or drug), peptide or polypeptide. In particular embodiments, the naked antibody expressed by a mammalian host cell may be glycosylated by the glycosylation machinery (e.g., glycosylase) of the host cell. In certain embodiments, the naked antibody is not glycosylated when expressed by a host cell that does not have its own glycosylation machinery (e.g., a glycosylase). In certain embodiments, the naked antibody is an intact antibody, while in other embodiments, the naked antibody is an antigen binding fragment of an intact antibody, such as a Fab antibody.
The term "conjugated antibody" herein refers to an antibody, which may be monoclonal, chimeric, humanized or human, that may be associated with a pharmaceutically acceptable carrier or diluent.
The term "diabody" herein refers to a bivalent, bispecific antibody that can bind to different epitopes on the same or different antigens.
As used herein, the term "percent (%) sequence identity" refers to the percentage of amino acid (or nucleotide) residues of a candidate sequence that are identical to amino acid (or nucleotide) residues of a reference sequence after aligning the sequences and introducing gaps, if desired, for maximum percent sequence identity (e.g., gaps may be introduced in one or both of the candidate and reference sequences for optimal alignment, and non-homologous sequences may be ignored for comparison purposes). For the purpose of determining percent sequence identity, the alignment may be accomplished in a variety of ways well known to those skilled in the art, for example using publicly available computer software such as BLAST, ALIGN, or Megalign (DNASTAIi) software. One skilled in the art can determine appropriate parameters for measuring the alignment, including any algorithm that requires maximum alignment over the full length of the sequences being compared. For example, a reference sequence for comparison to a candidate sequence may show that the candidate sequence exhibits 50% -100% sequence identity over the entire length of the candidate sequence or over selected portions of consecutive amino acid (or nucleotide) residues of the candidate sequence. The length of the candidate sequences aligned for comparison purposes may be, for example, at least 30% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%) of the length of the reference sequence. When a position in the candidate sequence is occupied by the same amino acid (or nucleotide) residue as the corresponding position in the reference sequence, then the molecules are identical at that position.
The term "conserved amino acids" herein generally refers to amino acids belonging to the same class or having similar characteristics (e.g., charge, side chain size, hydrophobicity, hydrophilicity, backbone conformation, and rigidity). The following six groups are examples of amino acids that are considered conservative substitutions for each other:
1) Alanine (a), serine (S), threonine (T);
2) Aspartic acid (D), glutamic acid (E);
3) Asparagine (N), glutamine (Q);
4) Arginine (R), lysine (K), histidine (H);
5) Isoleucine (I), leucine (L), methionine (M), valine (V); and
6) Phenylalanine (F), tyrosine (Y), tryptophan (W).
As used herein, the term "specific binding" refers to a binding reaction that determines the presence of an antigen in a heterogeneous population of proteins and other biomolecules that are specifically recognized, for example, by antibodies or antigen-binding fragments thereof. An antibody or antigen binding fragment thereof that specifically binds to an antigen will bind to the antigen with a KD of less than 100 nM. Antibodies or antigen binding fragments thereof that specifically bind to an antigen will bind to the antigen with a KD of up to 100nM (e.g., between 1pM and 100 nM) Antibodies or antigen binding fragments thereof that do not show specific binding to a particular antigen or epitope thereof will show greater than 100nM (e.g., greater than 500nM, 1 μΜ, 100 μΜ, 500 μΜ, or 1 mM) of the particular antigen or epitope thereof, antibodies that specifically immunoreact with a particular protein or carbohydrate can be selected Using a variety of immunoassay formats, e.g., solid phase ELISA immunoassays are routinely used to select Antibodies that specifically immunoreact with a protein or carbohydrate see Harlow & Lane, antibodies ALaboratory Manual, cold Spring Harbor Press, newYork (1988) and Harlow & Lane, using Antibodies A Laboratory Manual, 84, newYork (1999), which describe immunoassay formats and conditions that can be used to determine specific immunoreactivity.
As used herein, the term "antibody conjugate" refers to a conjugate body/conjugate formed by the chemical bonding of an antibody molecule to another molecule, either directly or through a linker. Such as an antibody-drug conjugate (ADC), wherein the drug molecule is said another molecule.
The term "Chimeric Antigen Receptor (CAR)" herein refers to a recombinant protein comprising at least (1) an extracellular antigen binding domain, such as a variable heavy or light chain of an antibody, (2) a transmembrane domain that anchors the CAR into immune effector cells, and (3) an intracellular signaling domain. In certain embodiments, the extracellular antigen-binding domain of the CAR comprises an scFv. The scFv may be derived from the variable region of a fusion antibody. Alternatively or additionally, the scFv may be derived from Fab's (rather than antibodies, e.g. obtained from a Fab library). In certain embodiments, the scFv is fused to a transmembrane domain and then to an intracellular signaling domain.
The term "nucleic acid" herein includes any compound and/or substance comprising a polymer of nucleotides. Each nucleotide consists of a base, in particular a purine or pyrimidine base (i.e. cytosine (C), guanine (G), adenine (a), thymine (T) or uracil (U)), a sugar (i.e. deoxyribose or ribose), and a phosphate group. In general, a nucleic acid molecule is described by a sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is usually represented as 5 'to 3'. In this context, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), in particular messenger RNA (mRNA), synthetic forms of DNA or RNA, and polymers comprising a mixture of two or more of these molecules. The nucleic acid molecule may be linear or circular. Furthermore, the term nucleic acid molecule includes both sense and antisense strands, as well as single-and double-stranded forms. Furthermore, the nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases having derivatized sugar or phosphate backbone bonded or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules suitable as vectors for direct expression of the antibodies of the invention in vitro and/or in vivo, e.g., in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors may be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA vector and/or expression of the encoded molecule, so that mRNA can be injected into a subject to produce antibodies in vivo (see, e.g., stadler et al, nature Medicine 2017,published online2017, 6-month 12, doi:10.1038/nm.4356 or EP 2 101 823B 1).
As used herein, the term "vector" includes nucleic acid vectors, such as DNA vectors (e.g., plasmids), RNA vectors, viruses, or other suitable replicons (e.g., viral vectors). A variety of vectors have been developed for delivering polynucleotides encoding exogenous proteins into prokaryotic or eukaryotic cells. The expression vectors of the invention contain polynucleotide sequences and additional sequence elements, for example, for expressing proteins and/or integrating these polynucleotide sequences into the genome of mammalian cells. Certain vectors that may be used to express the antibodies and antibody fragments of the invention include plasmids containing regulatory sequences (e.g., promoter and enhancer regions) that direct transcription of genes. Other useful vectors for expressing antibodies and antibody fragments contain polynucleotide sequences that enhance the translation rate of these genes or improve the stability or nuclear export of mRNA produced by gene transcription. These sequence elements include, for example, 5 'and 3' untranslated regions, internal Ribosome Entry Sites (IRES) and polyadenylation signal sites, in order to direct efficient transcription of genes carried on expression vectors. The expression vectors of the invention may also contain polynucleotides encoding markers for selecting cells containing such vectors. Examples of suitable markers include genes encoding antibiotic (e.g., ampicillin, chloramphenicol, kanamycin, or nociceptin) resistance.
The term "host cell" as used herein refers to a cell into which exogenous nucleic acid has been introduced, and includes the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include primary transformed cells and progeny derived therefrom, regardless of the number of passages. The progeny may not be exactly identical in nucleic acid content to the parent cell, but may comprise the mutation. Included herein are mutant progeny that have the same function or biological activity as screened or selected in the initially transformed cells.
The term "pharmaceutical composition" herein refers to a formulation which exists in a form which allows for the biological activity of the active ingredient contained therein to be effective and which does not contain additional ingredients which have unacceptable toxicity to the subject to whom the pharmaceutical composition is administered.
The term "effective amount" herein refers to an amount of a therapeutic agent that is effective to prevent or ameliorate a disease condition or progression of the disease when administered alone or in combination with another therapeutic agent to a cell, tissue or subject. An "effective amount" also refers to an amount of a compound that is sufficient to alleviate symptoms, such as treating, curing, preventing or alleviating a related medical condition, or an increase in the rate of treating, curing, preventing or alleviating such conditions. When an active ingredient is administered to an individual alone, a therapeutically effective dose is referred to as the ingredient alone. When a combination is used, a therapeutically effective dose refers to the combined amounts of the active ingredients that produce a therapeutic effect, whether administered in combination, sequentially or simultaneously.
The term "suitable conditions" herein refers to conditions suitable for culturing a variety of host cells, including eukaryotic cells and prokaryotic cells.
The term "cancer" herein refers to or describes a physiological condition in a mammal that is typically characterized by unregulated cell growth. Included in this definition are benign and malignant cancers.
The term "tumor" herein refers to all neoplastic (neoplastic) cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer" and "tumor" are not mutually exclusive when referred to herein.
The term "antitumor agent" herein refers to an antitumor drug, which is a class of drugs for treating tumor diseases, and there are chemotherapeutic drugs, biological agents, etc.
Advantageous effects
Compared with the prior art, the technical scheme of the invention has at least one of the following beneficial effects:
1. compared with alpaca antibodies, the humanized antibody disclosed by the invention has the capability of combining with HSA, reduces immunogenicity while ensuring that the molecular stability and biological functions meet the requirements, and is beneficial to reducing the immune rejection risk of human subjects in use.
2. The humanized antibody of the invention shows good binding capacity with human, mouse and/or monkey serum albumin, which is beneficial to improving the treatment effect and/or developing preclinical animal experiments.
3. In terms of binding to human, murine and/or monkey serum albumin, it was unexpectedly found that the humanized antibodies of the invention are comparable or superior to their parent alpaca antibodies, over conventional humanized antibodies (the binding capacity of conventional humanized antibodies is reduced 2-3 fold compared to the parent antibodies).
Drawings
Unless defined otherwise herein, scientific and technical terms used in connection with the present invention shall have the meaning as understood by one of ordinary skill in the art.
FIG. 1 is an electrophoresis diagram of a nested PCR two-round amplification alpaca nanobody gene sequence, and lane M is a protein Marker band.
FIG. 2 is a diagram showing the number of transformants, wherein the left panel shows the number of transformants in bacterial liquid diluted 1000-fold, and the right panel shows the number of transformants diluted 10000-fold.
FIG. 3 is an electrophoretogram of colony PCR for the insertion rate detection of clones after electrotransformation, lane M being a protein Marker band.
FIG. 4 shows SDS-PAGE and SEC-HPLC purity assays after purification of HSA control antibodies.
FIG. 5 is a chart showing the SEC-HPLC method for detecting the purity of the alpaca VHH-Fc antibody of the present invention.
Detailed Description
The invention will be further described with reference to specific embodiments, and advantages and features of the invention will become apparent from the description. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The present embodiments are merely examples and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes and substitutions of details and forms of the technical solution of the present invention may be made without departing from the spirit and scope of the present invention, but these changes and substitutions fall within the scope of the present invention.
Example 1 screening for human serum albumin nanobodies
1.1 detection of immune and serum titers of alpaca
1 Alpaca (Alpaca) aged 1.5-3 years was selected, and 10ml was collected before immunization and left as negative control serum. The first immunization was performed by subcutaneous injection after 0.5mg of human serum albumin (Chengdu Rong, national drug standard S10940024) was thoroughly mixed with Freund' S adjuvant (Sigma, F5881) using the prime dose doubling principle. Three weeks later, a second immunization was performed, 0.25mg of protein was mixed with Freund's adjuvant and then subcutaneously immunized, and serum was taken one week later for potency measurement. Third immunization 0.25mg protein was mixed with Freund's adjuvant and then subcutaneously immunized, and serum was taken one week later for potency measurement. To increase the probability of cross-binding of human and mouse, the fourth immunization was performed with a 0.25mg each of human serum albumin and mouse serum albumin (Alpha diagnostic, ALB 14-N-10) mixed together, and a 0.5mg challenge immunization after one week. Antibody titers and specificities of serum against human serum albumin were measured by enzyme-linked immunosorbent assay (ELISA), and the results of serum titers of alpaca from the third and fourth immunizations are shown in Table 1, where the data are OD450nm values.
TABLE 1 ELISA detection of serum antibody titers of alpaca after immunization with human serum albumin
1.2 construction of anti-human serum Albumin nanobody library
The peripheral blood of alpaca after the third and fourth immunizations was collected for 50mL and PBMCs were isolated using lymphocyte separation fluid. Total RNA after three and four immunizations was extracted with RNAiso Plus reagent (Takara, 9108/9109). According to the reverse transcription kit PrimeScript TM II 1st Strand cDNA Synthesis Kit (Takara, 6210A) protocol, 5. Mu.g RNA was co-transcribed. The nanobody (VHH) fragments were amplified using nested PCR with cDNA as template. FIG. 1 shows the results of the first and second rounds of amplification of VHH fragments, showing that the size of the target band after the first round of amplification is about 750bp and that after the second round of amplification is about 500bp. After purification of the PCR product, it was ligated into phage display vector pComb3Xss (Chengdu Parker, P001) using restriction enzyme SfiI (NEB, R0123L). Then the ligation product was electrotransformed into TG1 competent cells, 10 times of shock transformation were performed, 1mL of 2YT medium (Industry, A507019-0250) was added to the cuvette immediately after the shock for resuscitation, the shock product was aspirated and the cuvette was washed with 2YT medium (Industry, A507019-0250) to obtain 100mL in total TG1 cells resuscitate the product. Recovering TG1 cells at 37deg.C and 180rpm for 45min, and gradient diluting 100ml TG1 bacteria solution for 10 min 3 Double sum 10 4 The number of transformants in the nanobody library was determined by doubling and 10 3 And 10 4 The diluted TG1 bacterial liquid is coated on a 90mm plate, the rest bacterial liquid is centrifuged, 8mL 2YT culture medium (Producer, A507019-0250) is added for resuspension, and the bacterial liquid is coated on 8 200mm plates. The results of the number of transformants in gradient dilution are shown in FIG. 2, in the panel "NB116 10" for determining the number of transformants in the nanobody pool -4 "200 clones in total, calculated as the size of the pool size was 2.0X10 9 . FIG. 3 shows that 48 clones were randomly picked from the pool of transformants number titer plates for identification and the DNA band size of the protein of interest was approximately 500bp. The results showed that 48 clones were all positive, indicating an insertion rate of 100%.
First round amplification of the upstream primer LD-F:
first round amplification of the downstream primer CH2-R:
the second round of amplification upstream Primer is Primer F:
second round amplification of the downstream Primer R1:
second round amplification of the downstream Primer R2:
1.3 panning against human serum Albumin nanobody
Diluting the antigen molecule (Dongren chemical, LK 55) marked by biotin with carbonate buffer solution with pH value of 9.6 to a final concentration of 5 mug/mL, adding 100 mug/hole into enzyme-labeled hole, and coating at 4 ℃ overnight; the next day, 300. Mu.L of 3% OVA-PBS blocking solution was added to each well, after blocking for 1h at 37℃100. Mu.L of phage library was added and incubated for 1h at 37 ℃; followed by 6 washes with PBST and 2 washes with PBS to wash away unbound phage. Finally, 100. Mu.L Gly-HCl eluent is added, and incubated at 37 ℃ for 8min, and specifically bound phage is eluted. The eluted phage is continuously infected with TG1 strain, and positive phage is continuously enriched in the process of panning through multiple rounds of panning so as to screen out nano antibodies with good specificity and high affinity.
1.4 screening of specific individual Positive clones by phage ELISA
After panning, 96 clones were randomly selected for inoculation in 2YT medium (Bio, a 507019-0250), followed by 1:20 was added to M13K07 phage (NEB, N0315S) and incubated at 37℃for 45min. The next day the culture supernatant was taken for monoclonal ELISA identification. The albumin antigen of target molecule human, mouse and monkey was diluted with carbonate buffer solution with pH 9.6 to a final concentration of 2. Mu.g/mL, added to the enzyme-labeled wells at 100. Mu.L/well, and coated overnight at 4 ℃. Then 35. Mu.L of phage culture supernatant and 1:1000 dilution of horseradish peroxidase-labeled anti-M13 antibody (Proprietary, 11973-MM 05T-H) were added to each well, washed, followed by addition of TMB chromogenic solution (Soxhobao, PR 1200) for development and densitometry at 450 nm. Positive clones were selected for simultaneous binding to human, murine, and monkey albumin (Abcam, ab 184894) for sequencing. Through multiple rounds of optimization and screening, 3 positive clones capable of simultaneously recognizing human, mouse and monkey albumin are obtained and named sdAb-76, sdAb-220 and sdAb-350 respectively. The CDRs of the sequences are analyzed by KABAT, chothia or IMGT software respectively, and the corresponding sequence information is shown in the following tables 2-3, wherein the table 2 shows the antibody sequences expressed by amino acids of 3 alpaca nanobody molecules, and the table 3 shows the results of IMGT, kabat and Chothia analysis of the CDRs of the 3 alpaca nanobody molecules.
TABLE 2 amino acid specific sequence information of anti-HSA alpaca nanobody
TABLE 3 analysis of CDRs specific sequence information of HSA alpaca nanobody by IMGT, KABAT and Chothia software
Example 2 preparation of control antibodies
2.1 Recombinant expression of HSA control antibodies
The control antibody used in the present invention was obtained from the block structure of Vobarilizumab with anti-HSA antibody function (the sequence is from patent publication WO2018/029182 by Ablynx Co., ltd.) and the sequence is shown in Table 4 below. The control antibody variable region sequence was cloned into pTT5 vector (Youbao organism, VT 2202) with human Fc hinge region and constant region sequences, fusion expressed with the antibody constant region to form VHH-Fc expression sequence, and plasmids were prepared. Antibody plasmids were transiently transfected into Expi293F (Gibco, A14527) cells by PEI (Polysciences, 24765-1), and supernatants were collected after 7 days of culture and antibody purified as in example 2.2. The resulting HSA control antibody was designated aHSA-Fc. As shown in FIG. 4, the aHSA-Fc antibody had a molecular weight of about 40kD in the reduced state and about 80kD in the non-reduced state, and was detected to have a purity of 95% by the SEC-HPLC method.
TABLE 4 control antibody sequence listing
Remarks: * Represents a stop codon.
2.2 Protein A affinity chromatography purification Fc tag nano antibody
The cell culture supernatant expressing the antibody was first harvested by high-speed centrifugation. The protein A (Bognon, AA 0273) protein column was washed 3-5 column volumes with 0.1M NaOH and then 3-5 column volumes with pure water. The column was equilibrated 3-5 column volumes using a 1 XPBS (pH 7.4) buffer system as an equilibration buffer. The cell supernatants were loaded for binding at low flow rates, the flow rates were controlled to allow retention times of about 1min or longer, and after binding, the column was washed 3-5 column volumes with 1 XPBS (pH 7.4) until UV absorbance fell back to baseline. Sample elution was performed using 50mM citric acid/sodium citrate (pH 3.0-3.5) buffer, elution peaks were collected according to UV detection, and the eluted product was rapidly adjusted to pH 5-6 using 1M Tris-HCl (pH 8.0) for buffer storage. For the eluted product, solution displacement may be performed by methods well known to those skilled in the art, such as ultrafiltration concentration using an ultrafiltration tube and solution displacement to a desired buffer system, or desalting using a molecular exclusion column such as G-25 to a desired buffer system, or removing the polymer component in the eluted product using a high resolution molecular exclusion column such as Superdex 200 to increase the sample purity.
Example 3 recombinant nanobody expression purification and affinity detection
3.1 expression purification of recombinant nanobodies
The obtained alpaca nanobody sequences were cloned onto eukaryotic expression vectors pTT5 (Youbao organism, VT2202, fc sequences see Table 4) with Fc tags respectively, PEI (Polysciences, 24765-1) transiently transferred the Expi293F cells (Gibco, A14527), the supernatants were collected after 6 days of culture, and the antibodies with Fc tags eluted from the protein A chromatographic column were collected by affinity purification of protein A protein (Boguron, AA 0273) to obtain the corresponding recombinant nanobodies (VHH-Fc) named ch-sdAb-76, ch-sdAb-220 and ch-sdAb-350 respectively. The antibodies obtained by purification are detected by SEC-HPLC method, and the result of one-step purification of Protein A is shown in figure 5, which shows that the purity of three antibodies is more than 95%.
3.2 affinity assay of recombinant nanobodies
The binding strength of the antibodies to the antigen was detected using a BIAcore 8K instrument using the Protein A capture method. First, protein A was immobilized on CM4 chip (manufacturer: GE, cat. No. BR-1005-34) by amino coupling method, after mixing NHS and EDC with HBS-EP+pH7.4 as mobile phase according to the direction of Amine Coupling Kit kit (manufacturer: GE, cat. No. BR 100633), the chip was activated for about 600 seconds, protein A was diluted to 50. Mu.g/mL with 10mM sodium acetate pH4.5, injected for 600 seconds, and finally the remaining activation sites were blocked with ethanolamine. Then, determining the affinity of the antibody and the antigen by adopting a multi-cycle dynamics method, in each cycle, firstly capturing the antibody to be detected by using a Protein A chip, then injecting antigen Protein with single concentration, recording the combination and dissociation processes of the antibody and the antigen Protein, and finally finishing chip regeneration by using Glycine pH1.5, wherein the mobile phase is HBS-EP+ (10mM HEPES,150mM NaCl,3mM EDTA, 0.05%surfactant P20), the flow rate is 30 mu L/min, the regeneration time is 30s, and the detection temperature is 25 ℃; finally, the data were analyzed according to the 1:1 binding model, fitting antibody antigen binding kinetic parameters including binding rate constant ka, dissociation rate constant KD, equilibrium dissociation constant KD, maximum binding signal Rmax.
The binding rate (Ka), dissociation rate (Kd) and binding affinity (Kd) of the 3 recombinant nanobodies to human, mouse and monkey albumin are shown in table 5. Wherein, the affinity of the three recombinant nano antibodies against human, mouse and monkey serum albumin is better than that of the control antibody aHSA-Fc (SEQ ID NO. 57), as shown in Table 5, the three recombinant nano antibodies are combined with human, mouse and monkey serum albumin, the affinity with human serum albumin is above 6.87E-09M, the affinity with monkey serum albumin is above 7.10E-09M, and the affinity with mouse serum albumin is above 1.04E-08M.
TABLE 5 SPR (biacore) detection of affinity of recombinant nanobodies to human, monkey, mouse serum albumin
EXAMPLE 4 nanobody humanization
4.1 humanized design of alpaca nanobody
The variable region germline gene database and MOE (Molecular Operating Environment ) software of human antibody in IMGT (http:// IMGT. Cines. FR) database are respectively selected and used as templates for heavy chain variable region germline genes with high homology with the nanobody, and CDRs sequences of the nanobody based on Kabat naming method are respectively transplanted into corresponding human templates to form variable region sequences with the sequence of 'FR 1-CDR1-FR2-CDR2-FR3-CDR3-FR 4'. Humanized templates of the sdAb-76 alpaca nanobody are IGHV3-23 x 04 and IGHJ2 x 01, and humanized templates of the sdAb-350 and sdAb-220 alpaca nanobody are IGHV3-23 x 04 and IGHJ3 x 01. CDRs of alpaca antibodies sdAb-76, sdAb-350 and sdAb-220 were transplanted into their humanized templates, respectively, and the amino acid sequences of the humanized templates and the amino acid sequences of the 3 humanized antibodies are shown in Table 6.
TABLE 6 amino acid specific sequence information of humanized templates and humanized antibodies
4.2 humanized antibody back mutation design of hu-sdAb76
The key amino acid in the FR region sequence of the hu-sdAb76 humanized antibody is subjected to back mutation to be the amino acid corresponding to the alpaca antibody, so that the original affinity is ensured, and details of mutation points after specific back mutation (the back mutation points are numbered in natural sequence) and specific amino acid sequences are shown in tables 7-8.
TABLE 7 mutation Point cases of the back mutation of hu-sdAb76 humanized antibodies
Antibody name Sequence numbering Mutation point
hu-sdAb76-H1 SEQ ID NO.7 Graft(IGHV3-23*04)+S30N,V37F,I70V,A97R,K98Q,W109R
hu-sdAb76-H2 SEQ ID NO.8 Graft(IGHV3-23*04)+S30N,V37F,I70V,A97R,K98Q,W109R,E1Q
hu-sdAb76-H3 SEQ ID NO.9 Graft(IGHV3-23*04)+S30N,V37F,I70V,A97R,K98Q,W109R,S75A
hu-sdAb76-H4 SEQ ID NO.10 Graft(IGHV3-23*04)+S30N,V37F,I70V,A97R,K98Q,W109R,Q82E
hu-sdAb76-H5 SEQ ID NO.11 Graft(IGHV3-23*04)+S30N,V37F,I70V,A97R,K98Q,W109R,S85N
hu-sdAb76-H6 SEQ ID NO.12 Graft(IGHV3-23*04)+S30N,V37F,I70V,A97R,K98Q,W109R,L114Q
Note that: graft (IGHV 3-23X 04) represents the implantation of alpaca antibody CDRs into human germline FR region sequences; A97R represents the mutation of A at position 97 of Graft (IGHV 3-23.04) to R, and so on.
TABLE 8 amino acid sequence of the back mutation of hu-sdAb76 humanized antibodies
Note that: the bolded parts represent mutation points
4.3 humanized antibody back mutation design of hu-sdAb220
The key amino acid in the FR region sequence of the hu-sdAb220 humanized antibody is subjected to back mutation to be the amino acid corresponding to the alpaca antibody, so that the original affinity is ensured, and details of mutation points after specific back mutation (the back mutation points are numbered in natural sequence) and specific amino acid sequences are shown in tables 9-10.
TABLE 9 mutation Point cases of the back mutation of the hu-sdAb220 humanized antibody
Note that: graft (IGHV 3-23X 04) represents the implantation of alpaca antibody CDRs into human germline FR region sequences; E1Q represents the mutation of E at position 1 of Graft (IGHV 3-23.04) to Q, and so on.
TABLE 10 amino acid sequence of the back mutation of hu-sdAb220 humanized antibodies
Note that: the bolded parts represent mutation points
4.4 humanized antibody back mutation design of hu-sdAb350
The key amino acids in the FR region sequence of the hu-sdAb350 humanized antibody are subjected to back mutation to amino acids corresponding to the alpaca antibody, so that the original affinity is ensured, and details of mutation points after specific back mutation (the back mutation points are numbered in natural sequence) and specific amino acid sequences are shown in tables 11-12.
TABLE 11 mutation Point cases of the back mutation of hu-sdAb350 humanized antibodies
Antibody name Sequence numbering Mutation point
hu-sdAb350-H1 SEQ ID NO.20 Graft(IGHV3-23*04)+A97S,K98I
hu-sdAb350-H2 SEQ ID NO.21 Graft(IGHV3-23*04)+A97S,K98I,W104S
hu-sdAb350-H3 SEQ ID NO.22 Graft(IGHV3-23*04)+V2L,A97S,K98I,W104S
hu-sdAb350-H4 SEQ ID NO.23 Graft(IGHV3-23*04)+T28A,A97S,K98I,W104S
hu-sdAb350-H5 SEQ ID NO.24 Graft(IGHV3-23*04)+V2L,T28A,A97S,K98I,W104S
hu-sdAb350-H6 SEQ ID NO.25 Graft(IGHV3-23*04)+R87K,A88P,A97S,K98I,W104S
hu-sdAb350-H7 SEQ ID NO.26 Graft(IGHV3-23*04)+V2L,T28A,S75A,A97S,K98I,W104S
hu-sdAb350-H8 SEQ ID NO.27 Graft(IGHV3-23*04)+V2L,T28A,R87K,A88P,A97S,K98I,W104S
Note that: graft (IGHV 3-23X 04) represents the implantation of alpaca antibody CDRs into human germline FR region sequences; A97S indicates that A at position 97 of Graft (IGHV 3-23.04) is mutated to S, and so on.
TABLE 12 amino acid sequence of the back mutation of hu-sdAb350 humanized antibodies
Note that: the bolded parts represent mutation points
EXAMPLE 5 expression purification and affinity determination of humanized nanobodies
5.1 expression purification of humanized nanobodies
After the humanized antibody variable region sequence is synthesized (completed by Shanghai, inc.) it is cloned into pTT5 vector (Youbao organism, VT 2202) with human Fc hinge region and constant region sequence, and fusion expressed with antibody constant region to form VHH-Fc expression sequence, and plasmid is prepared. Antibody plasmids were transiently transfected into Expi293F (Gibco, A14527) cells by PEI (Polysciences, 24765-1), and supernatants were collected after 7 days of culture and antibody purified as in example 2.2.
5.2 Affinity assay for hu-sdAb76 humanized antibodies
After purification of the expression of the various hu-sdAb76 humanized antibodies finally obtained, the affinity of the humanized antibodies to human, monkey and mouse serum albumin was determined separately according to the method of example 3.2, and the specific affinity values are shown in table 13. Through back mutation, the humanized antibody hu-sdAb76-H4 maintains an affinity level with the chimeric antibody, substantially comparable to the chimeric control antibody.
TABLE 13 SPR (biacore) detection of affinity of hu-sdAb76 humanized antibodies to human, monkey, mouse serum albumin
Antibody name Antigens ka(1/Ms) kd(1/s) KD(M)
ch-sdAb-76 Human serum albumin 5.12E+05 1.86E-04 3.64E-10
ch-sdAb-76 Monkey serum albumin 5.27E+05 1.40E-03 2.65E-09
ch-sdAb-76 Mouse serum albumin 1.30E+05 5.78E-04 4.44E-09
hu-sdAb76-H1 Human serum albumin 1.47E+05 2.53E-04 1.71E-09
hu-sdAb76-H1 Monkey serum albumin 3.25E+05 6.22E-03 1.92E-08
hu-sdAb76-H1 Mouse serum albumin No binding No binding No binding
hu-sdAb76-H2 Human serum albumin 1.37E+05 2.35E-04 1.72E-09
hu-sdAb76-H2 Monkey serum albumin 2.24E+05 4.02E-03 1.80E-08
hu-sdAb76-H2 Mouse serum albumin 4.00E+04 3.57E-04 8.92E-09
hu-sdAb76-H3 Human serum albumin 1.48E+05 2.89E-04 1.95E-09
hu-sdAb76-H3 Monkey serum albumin 2.52E+05 5.23E-03 2.08E-08
hu-sdAb76-H3 Mouse serum albumin 3.30E+04 4.11E-04 1.25E-08
hu-sdAb76-H4 Human serum albumin 4.74E+05 1.70E-04 3.60E-10
hu-sdAb76-H4 Monkey serum albumin 4.45E+05 1.59E-03 3.58E-09
hu-sdAb76-H4 Mouse serum albumin 7.50E+04 6.82E-04 9.09E-09
hu-sdAb76-H5 Human serum albumin 1.48E+05 2.99E-04 2.02E-09
hu-sdAb76-H5 Monkey serum albumin 2.18E+05 5.01E-03 2.30E-08
hu-sdAb76-H5 Mouse serum albumin 2.26E+04 5.41E-04 2.39E-08
hu-sdAb76-H6 Human serum albumin 1.49E+05 3.12E-04 2.10E-09
hu-sdAb76-H6 Monkey serum albumin 2.31E+05 5.43E-03 2.36E-08
hu-sdAb76-H6 Mouse serum albumin 2.68E+04 5.02E-04 1.87E-08
5.3 Affinity assay for hu-sdAb220 humanized antibodies
After purification of the finally obtained humanized antibodies of hu-sdAb220 expression, the affinity of the antibodies to human, monkey, mouse, rat serum albumin, respectively, was determined according to the method of example 3.2, and the specific affinity values are shown in table 14. The results show that: all hu-sdAb220 humanized antibodies remained at levels comparable to the chimeric control antibodies through back-mutation, binding to human serum albumin and monkey serum albumin. However, the hu-sdAb220 humanized antibodies have weaker binding to mouse serum albumin than chimeric antibodies. Presumably, this is due to the fact that the binding epitope of the hu-sdAb220 nanobody to mouse albumin is more offset from the CDR regions, and more FR regions are involved in protein binding.
TABLE 14 SPR (biacore) detection of affinity of hu-sdAb220 humanized antibodies to human, monkey, mouse serum albumin
Antibody name Antigens ka(1/Ms) kd(1/s) KD(M)
ch-sdAb-220 Human serum albumin 2.47E+05 9.98E-04 4.04E-09
ch-sdAb-220 Monkey serum albumin 2.80E+05 1.98E-03 7.10E-09
ch-sdAb-220 Mouse serum albumin 5.34E+04 2.20E-04 4.13E-09
hu-sdAb220-H1 Human serum albumin 3.70E+05 1.26E-03 3.40E-09
hu-sdAb220-H1 Monkey serum albumin 3.61E+05 2.16E-03 5.98E-09
hu-sdAb220-H1 Mouse serum albumin 2.58E+04 4.39E-04 1.70E-08
hu-sdAb220-H2 Human serum albumin 3.62E+05 1.34E-03 3.70E-09
hu-sdAb220-H2 Monkey serum albumin 3.54E+05 2.27E-03 6.41E-09
hu-sdAb220-H2 Mouse serum albumin 2.16E+04 3.86E-04 1.78E-08
hu-sdAb220-H3 Human serum albumin 3.79E+05 1.34E-03 3.54E-09
hu-sdAb220-H3 Monkey serum albumin 3.65E+05 2.26E-03 6.19E-09
hu-sdAb220-H3 Mouse serum albumin 2.82E+04 5.35E-04 1.89E-08
hu-sdAb220-H4 Human serum albumin 3.68E+05 1.31E-03 3.57E-09
hu-sdAb220-H4 Monkey serum albumin 3.65E+05 2.22E-03 6.08E-09
hu-sdAb220-H4 Mouse serum albumin 2.39E+04 3.08E-04 1.29E-08
hu-sdAb220-H5 Human serum albumin 3.68E+05 1.11E-03 3.02E-09
hu-sdAb220-H5 Monkey serum albumin 3.57E+05 1.92E-03 5.38E-09
hu-sdAb220-H5 Mouse serum albumin 2.53E+04 3.01E-04 1.19E-08
hu-sdAb220-H6 Human serum albumin 3.84E+05 1.17E-03 3.05E-09
hu-sdAb220-H6 Monkey serum albumin 3.72E+05 2.04E-03 5.49E-09
hu-sdAb220-H6 Mouse serum albumin 3.10E+04 4.36E-04 1.41E-08
hu-sdAb220-H7 Human serum albumin 3.64E+05 1.30E-03 3.56E-09
hu-sdAb220-H7 Monkey serum albumin 3.53E+05 2.24E-03 6.35E-09
hu-sdAB220-H7 Mouse serum albumin 2.34E+04 3.64E-04 1.55E-08
Considering the closer homology between mice and rats, we further determined the affinity of antibodies to rat serum albumin according to the method of example 3.2, and the specific affinity values are shown in Table 15. The results show that: the affinity of the control antibody aHSA-Fc for rat serum albumin was very weak, only 5.00E-07M. The hu-sdAb220 was back mutated, and all humanized antibodies retained levels of affinity for rats comparable to chimeric antibodies.
TABLE 15 SPR (biacore) detection of affinity of hu-sdAb220 humanized antibodies to rat serum albumin
Antibody name Antigens ka(1/Ms) kd(1/s) KD(M)
ch-sdAb-220 Rat serum albumin 1.22E+06 3.31E-02 2.71E-08
hu-sdAb220-H1 Rat serum albumin 1.05E+06 3.16E-02 3.01E-08
hu-sdAb220-H2 Rat serum albumin 1.20E+06 3.40E-02 2.84E-08
hu-sdAb220-H3 Rat serum albumin 1.12E+06 3.32E-02 2.95E-08
hu-sdAb220-H4 Rat serum albumin 1.20E+06 2.94E-02 2.46E-08
hu-sdAb220-H5 Rat serum albumin 1.20E+06 3.01E-02 2.51E-08
hu-sdAb220-H6 Rat serum albumin 1.11E+06 3.30E-02 2.97E-08
hu-sdAb220-H7 Rat serum albumin 8.35E+05 2.24E-02 2.68E-08
aHSA-Fc control Rat serum albumin 6.84E+04 3.42E-02 5.00E-07
5.4 Affinity assay for hu-sdAb350 humanized antibodies
After purification of the finally obtained humanized antibodies of the hu-sdAb350 expression, the affinity of the antibodies for human, monkey and mouse serum albumin, respectively, was determined according to the method of example 3.2, and the specific affinity values are shown in table 16. The results show that: the affinity of the humanized antibodies was maintained at a level comparable to that of the chimeric control antibody except for the hu-sdAb350-H1 by back-mutation.
TABLE 16 SPR (biacore) detection of affinity of hu-sdAb350 humanized antibodies to human, monkey, mouse serum albumin
Antibody name Antigens ka(1/Ms) kd(1/s) KD(M)
ch-sdAb-350 Human serum albumin 1.53E+05 1.05E-03 6.87E-09
ch-sdAb-350 Monkey serum albumin 1.50E+05 8.80E-04 5.85E-09
ch-sdAb-350 Mouse serum albumin 8.19E+04 8.56E-04 1.04E-08
hu-sdAb350-H1 Human serum albumin 1.16E+05 1.73E-02 1.50E-07
hu-sdAb350-H1 Monkey serum albumin 5.63E+05 1.45E-02 2.58E-08
hu-sdAb350-H1 Mouse serum albumin 4.74E+04 1.21E-03 2.55E-08
hu-sdAb350-H2 Human serum albumin 1.34E+05 7.90E-04 5.88E-09
hu-sdAb350-H2 Monkey serum albumin 1.28E+05 6.62E-04 5.18E-09
hu-sdAb350-H2 Mouse serum albumin 7.69E+04 1.17E-03 1.53E-08
hu-sdAb350-H3 Human serum albumin 1.40E+05 1.08E-03 7.70E-09
hu-sdAb350-H3 Monkey serum albumin 1.32E+05 9.29E-04 7.02E-09
hu-sdAb350-H3 Mouse serum albumin 7.65E+04 1.06E-03 1.39E-08
hu-sdAb350-H4 Human serum albumin 1.33E+05 7.39E-04 5.54E-09
hu-sdAb350-H4 Monkey serum albumin 1.27E+05 6.31E-04 4.95E-09
hu-sdAb350-H4 Mouse serum albumin 7.56E+04 1.17E-03 1.55E-08
hu-sdAb350-H5 Human serum albumin 1.42E+05 1.02E-03 7.14E-09
hu-sdAb350-H5 Monkey serum albumin 1.34E+05 8.89E-04 6.63E-09
hu-sdAb350-H5 Mouse serum albumin 7.65E+04 1.05E-03 1.37E-08
hu-sdAb350-H6 Human serum albumin 1.34E+05 7.72E-04 5.78E-09
hu-sdAb350-H6 Monkey serum albumin 1.26E+05 6.50E-04 5.14E-09
hu-sdAb350-H6 Mouse serum albumin 7.77E+04 1.17E-03 1.51E-08
hu-sdAb350-H7 Human serum albumin 1.37E+05 1.05E-03 7.68E-09
hu-sdAb350-H7 Monkey serum albumin 1.65E+05 9.82E-04 5.95E-09
hu-sdAb350-H7 Mouse serum albumin 7.66E+04 1.03E-03 1.34E-08
hu-sdAb350-H8 Human serum albumin 1.43E+05 1.02E-03 7.13E-09
hu-sdAb350-H8 Monkey serum albumin 1.35E+05 8.96E-04 6.63E-09
hu-sdAb350-H8 Mouse serum albumin 7.82E+04 1.03E-03 1.32E-08

Claims (24)

  1. An isolated nanobody or antigen-binding fragment that binds Human Serum Albumin (HSA), wherein the antibody or antigen-binding fragment comprises a combination of CDRs of a heavy chain variable region comprising: HCDR1, HCDR2 and HCDR3; the HCDR1, HCDR2 and HCDR3 have any sequence combination selected from the group consisting of:
    each of HCDR1, HCDR2 and HCDR3 is encoded according to a current analytical method of KABAT, chothia or IMGT;
    preferably, the substitution is a conservative amino acid substitution.
  2. The nanobody or antigen-binding fragment of claim 1, wherein,
    (1) The HCDR1, HCDR2 and HCDR3 are respectively shown as SEQ ID NO.31, 32 and 33;
    (2) The HCDR1, HCDR2 and HCDR3 are respectively shown as SEQ ID NO.34, 35 and 36;
    (3) The HCDR1, HCDR2 and HCDR3 are respectively shown as SEQ ID NO.37, 38 and 36;
    (4) The HCDR1, HCDR2 and HCDR3 are respectively shown as SEQ ID NO.39, 40 and 41;
    (5) The HCDR1, HCDR2 and HCDR3 are respectively shown as sequences shown in SEQ ID NO.42, 43 and 44;
    (6) The HCDR1, HCDR2 and HCDR3 are respectively shown as sequences shown in SEQ ID NO.45, 46 and 44;
    (7) The HCDR1, HCDR2 and HCDR3 are respectively shown as SEQ ID NO.47, 48 and 49;
    (8) The HCDR1, HCDR2 and HCDR3 are respectively shown as SEQ ID NO.50, 51 and 52;
    (9) The HCDR1, HCDR2 and HCDR3 are respectively shown as SEQ ID NO.53, 54 and 52; or alternatively, the first and second heat exchangers may be,
    (10) The HCDR1, HCDR2 and HCDR3 are a sequence combination having 1, 2, 3 or more amino acid insertions, deletions and/or substitutions compared to a sequence combination of (1) - (9) above; substitutions are preferred, more preferably conservative amino acid residue substitutions.
  3. The nanobody or antigen-binding fragment of any one of claims 1-2, wherein the framework region of the heavy chain variable region of the nanobody or antigen-binding fragment is derived from a human germline heavy chain, wherein:
    (1) The framework region sequence is derived from the combined sequence of human germline heavy chains IGHV3-23 x 04 and IGHJ2 x 01; which comprises SEQ ID NO:28, the FR1, FR2, FR3 regions of igfv 3-23 x 04 and SEQ ID NO:29 to igfj 2 x 01;
    (2) The framework region sequence is derived from the combined sequence of human germline heavy chains IGHV3-23 x 04 and IGHJ3 x 01; which comprises SEQ ID NO:28, the FR1, FR2, FR3 regions of igfv 3-23 x 04 and SEQ ID NO:30, the FR4 region of IGHJ3 x 01.
  4. The nanobody or antigen-binding fragment of claim 3, wherein the framework region of the heavy chain variable region of the nanobody or antigen-binding fragment further comprises one or more mutations selected from the group consisting of:
    (1) E1Q, S30N, V F, I70V, S3756A, Q82E, S85N, A97R, K98Q, W109R or L114Q; preferably including S30N, V37F, I70V, A97R, K98Q and W109R; or preferably S30N, V37F, I70V, A97R, K98Q, W109R and E1Q; or preferably comprises S30N, V37F, I70V, A97R, K98Q, W109R and S75A; or preferably S30N, V37F, I70V, A97R, K98Q, W109R and Q82E; or preferably comprises S30N, V37F, I70V, A97R, K98Q, W109R and S85N; or preferably comprises S30N, V37F, I70V, A97R, K98Q, W109R and L114Q;
    (2) E1Q, A23V, F V, T28D, S R, S6275A, R87K, A88F, T M, A97V, K98Q, W P or M116Q; preferably including E1Q, F27V, T D, S R, S75A, T91M, A97V, K98Q, W P and M116Q; preferably including F27V, T28D, S30R, S A, T91M, A97V, K3598Q, W111P and M116Q; preferably including F27V, T28D, S R, T91M, A97V, K98Q, W P and M116Q; preferably including F27V, T28D, S30R, R87K, A88F, T91M, A97V, K98Q, W P and M116Q; preferably including E1Q, A23V, F27V, T D, S30R, S A, A97V, K Q and W111P; preferably comprising a23V, F27V, T28D, S R, S30 6275A, A97V, K Q and W111P; preferably including F27V, T28D, S30R, R87K, A F, T91M, A97V, K Q and W111P;
    (3) V2L, T28A, S75A, R87K, A88P, A97S, K I or W104S; preferably comprising a97S and K98I; preferably comprising a97S, K I and W104S; preferably comprising V2L, A97S, K I and W104S; preferably including T28A, A97S, K I and W104S; preferably comprising V2L, T28A, A97S, K98I and W104S; preferably, R87K, A88P, A97S, K98I and W104S; preferably comprising V2L, T28A, S A, A97S, K98I and W104S; preferably V2L, T28A, R87K, A88P, A97S, K98I and W104S.
  5. The nanobody or antigen-binding fragment of any one of claims 1-4, wherein said nanobody or antigen-binding fragment comprises:
    (1) The variable region has a sequence shown in SEQ ID NO 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27;
    (2) An amino acid sequence having at least 90% identity, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity, to the sequence shown in (1) above; or alternatively, the first and second heat exchangers may be,
    (3) The framework regions of the nanobody or antigen-binding fragment have at least 90% identity, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity, to the framework regions of the amino acid sequences shown in SEQ ID NOs 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27.
  6. Nanobody or antigen-binding fragment according to any one of claims 1 to 5, characterized in that it binds to human serum albumin, monkey serum albumin and/or rat serum albumin; alternatively, the antibody or antigen binding fragment binds to human serum albumin, monkey serum albumin, and/or rat serum albumin with a dissociation constant (KD) of less than 1.00E-8M, 2.00E-8M, 3.00E-8M, 1.00E-9M, 2.00E-09M, 3.00E-9M, 4.00E-09M, 5.00E-09M, 6.00E-09M, 7.00E-09M, 8.00E-09M, 9.00E-09M, 1.00E-10M, 2.00E-10M, 3.00E-10M, 4.00E-10M, 5.00E-10M, 6.00E-10M, 7.00E-10M, 8.00E-10M, 9.00E-10M, 1.00E-11M, 2.00E-11M, 3.00E-11M, 4.00E-11M, 1.00E-10M, 2.00E-11M, 3.00E-12.00E-12M, 12.00E-12M, 3.00E-12.00E-12M;
    alternatively, the nanobody or antigen-binding fragment binds to mouse serum albumin or does not bind.
  7. The nanobody or antigen-binding fragment of any one of claims 1-6, wherein the antibody or antigen-binding fragment comprises the sequence of the constant region of any one of antibodies IgG1, igG2, igG3, igG4, igA, igM, igE, or IgD; preferably comprising the sequences of the constant regions of antibodies IgG1, igG2, igG3 or IgG 4.
  8. The nanobody or antigen-binding fragment of any one of claims 1-7, wherein the antibody or antigen-binding fragment further comprises an antibody constant region sequence in the absence of a CH1 fragment.
  9. The nanobody or antigen-binding fragment of any one of claims 1-8, wherein the antibody or antigen-binding fragment further comprises an antibody constant region sequence having CH2 and CH3 fragments, or wherein the antibody or antigen-binding fragment further comprises an antibody Fc region;
    the antibody constant region or antibody Fc region is linked to the antibody or antigen binding fragment with or without a linking peptide;
    alternatively, the antibody constant region or antibody Fc region is from a camelid, mouse, rat, rabbit, sheep or human;
    alternatively, the antibody constant region or antibody Fc region is from IgG, igA, igM, igD or IgE.
  10. The nanobody or antigen-binding fragment of any one of claims 1-9, wherein the antibody or antigen-binding fragment is:
    (1) A chimeric antibody or fragment thereof;
    (2) A humanized antibody or fragment thereof; or alternatively, the first and second heat exchangers may be,
    (3) A fully human antibody or fragment thereof;
    preferably, the antibody or antigen binding fragment is selected from the group consisting of monoclonal antibodies, polyclonal antibodies, natural antibodies, engineered antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), monovalent antibodies, multivalent antibodies, full length antibodies, antibody fragments, naked antibodies, conjugated antibodies, humanized antibodies, fully human antibodies, fab ', F (ab') 2, fd, fv, scFv, diabodies (diabodies), or single domain antibodies.
  11. The antibody or antigen-binding fragment of any one of claims 1-10, wherein the antibody or antigen-binding fragment is further conjugated to a therapeutic agent or tracer; preferably, the therapeutic agent is selected from the group consisting of a radioisotope, a chemotherapeutic agent or an immunomodulator, and the tracer is selected from the group consisting of a radiocontrast agent, a paramagnetic ion, a metal, a fluorescent label, a chemiluminescent label, an ultrasound contrast agent or a photosensitizer.
  12. A multispecific antigen-binding molecule comprising a first antigen-binding moiety comprising the antibody or antigen-binding fragment of any one of claims 1-11, and a second antigen-binding moiety that specifically binds to an antigen other than HSA or to an HSA epitope different from the first antigen-binding moiety;
    preferably, the additional antigen is selected from the group consisting of CD2, CD3, CD4, CD5, CD7, CD8, CD11a, CD11b, CD18, CD19, CD20, CD23, CD25, CD33, CD38, CD40, CD45, CDW52, CD69, CD70, CD134 (OX 40), ICOS, BCMP7, CD137, CD27L, CDCP1, DPCR1, dudulin2, FLJ20584, FLJ40787, HEK2, KIAA0634, KIAA0659, KIAA1246, KIAA1455, LTBP2, LTK, MAL2, MRP2, MSLN;
    Preferably, the multispecific antigen-binding molecule is a bispecific, trispecific or tetraspecific antibody.
  13. A Chimeric Antigen Receptor (CAR) comprising at least an extracellular antigen-binding domain comprising the HSA antibody or antigen-binding fragment of any one of claims 1-11, a transmembrane domain, and an intracellular signaling domain.
  14. An immune effector cell comprising the chimeric antigen receptor of claim 13 or a nucleic acid fragment encoding the chimeric antigen receptor of claim 13;
    preferably, the immune effector cell is selected from T cells, NK cells (natural killer cell), NKT cells (natural killer T cell), monocytes, macrophages, dendritic cells or mast cells; the T cells may be selected from inflammatory T cells, cytotoxic T cells, regulatory T cells (tregs) or helper T cells;
    preferably, the immune effector cell is an allogeneic immune effector cell or an autoimmune cell.
  15. An isolated nucleic acid molecule encoding the nanobody, antigen-binding fragment, or any combination thereof of any of claims 1-11, the multispecific antigen-binding molecule of claim 12, or the chimeric antigen receptor of claim 13.
  16. An expression vector comprising the isolated nucleic acid molecule of claim 15.
  17. An isolated host cell comprising the isolated nucleic acid molecule of claim 15, or the expression vector of claim 16; preferably, the host cell is a eukaryotic cell or a prokaryotic cell; more preferably, the host cell is derived from a mammalian cell, a yeast cell, an insect cell, escherichia coli and/or bacillus subtilis; more preferably, the host cell is selected from the group consisting of an Expi293F cell.
  18. A method of making the antibody or antigen-binding fragment of any one of claims 1-11 or the multispecific antigen-binding molecule of claim 12, wherein the host cell of claim 17 is cultured under suitable conditions and the antibody or antigen-binding fragment or multispecific antigen-binding molecule is isolated.
  19. A method of making the immune effector cell of claim 14, comprising introducing into an immune effector cell a nucleic acid fragment encoding the CAR of claim 13, optionally further comprising activating the immune effector cell to express the CAR of claim 13.
  20. A pharmaceutical composition comprising the antibody or antigen-binding fragment of any one of claims 1-11, the multispecific antigen-binding molecule of claim 12, the immune effector cell of claim 14, the isolated nucleic acid molecule of claim 15, the expression vector of claim 16, the cell of claim 17, or the product of the method of claim 18 or 19; preferably, the composition further comprises a pharmaceutically acceptable carrier, diluent or adjuvant; preferably, the pharmaceutical composition further comprises an additional anti-tumor agent.
  21. Use of the antibody or antigen binding fragment of any one of claims 1-11, the multispecific antigen-binding molecule of claim 12, the immune effector cell of claim 14, the isolated nucleic acid molecule of claim 15, the expression vector of claim 16, the cell of claim 17, or the product prepared by the method of claim 18 or 19, or the pharmaceutical composition of claim 20, in the manufacture of a medicament for the prevention and/or treatment of a neoplastic disease or inflammatory disease;
    preferably, the neoplastic disease is preferably melanoma, clear cell sarcoma, head and neck cancer, bladder cancer, breast cancer, colon cancer, ovarian cancer, endometrial cancer, gastric cancer, pancreatic cancer, kidney cancer, prostate cancer, salivary gland cancer, lung cancer, liver cancer, skin cancer, or brain cancer;
    preferably, the inflammatory disease is preferably multiple sclerosis, psoriasis, myasthenia gravis, uveitis, systemic lupus erythematosus, or rheumatoid arthritis.
  22. A method of preventing and/or treating a neoplastic disease or inflammatory disease comprising administering to a patient in need thereof an effective amount of the antibody or antigen binding fragment of any one of claims 1-11, the multispecific antigen-binding molecule of claim 12, the immune effector cell of claim 14, the isolated nucleic acid molecule of claim 15, the expression vector of claim 16 or the product of the method of claim 18 or 19, or the pharmaceutical composition of claim 20;
    Preferably, the neoplastic disease is preferably melanoma, clear cell sarcoma, head and neck cancer, bladder cancer, breast cancer, colon cancer, ovarian cancer, endometrial cancer, gastric cancer, pancreatic cancer, kidney cancer, prostate cancer, salivary gland cancer, lung cancer, liver cancer, skin cancer, or brain cancer;
    preferably, the inflammatory disease is preferably multiple sclerosis, psoriasis, myasthenia gravis, uveitis, systemic lupus erythematosus, or rheumatoid arthritis.
  23. The antibody or antigen binding fragment of any one of claims 1-11, the multispecific antigen-binding molecule of claim 12, the immune effector cell of claim 14, the isolated nucleic acid molecule of claim 15, the expression vector of claim 16, the cell of claim 17, or the product made by the method of claim 18 or 19, or the pharmaceutical composition of claim 20, for use in and/or in the treatment of a neoplastic disease or inflammatory disease;
    preferably, the neoplastic disease is preferably melanoma, clear cell sarcoma, head and neck cancer, bladder cancer, breast cancer, colon cancer, ovarian cancer, endometrial cancer, gastric cancer, pancreatic cancer, kidney cancer, prostate cancer, salivary gland cancer, lung cancer, liver cancer, skin cancer, or brain cancer;
    Preferably, the inflammatory disease is preferably multiple sclerosis, psoriasis, myasthenia gravis, uveitis, systemic lupus erythematosus, or rheumatoid arthritis.
  24. A kit comprising the antibody or antigen-binding fragment thereof of any one of claims 1-11, the multispecific antigen-binding molecule of claim 12, the immune effector cell of claim 14, the isolated nucleic acid molecule of claim 15, the expression vector of claim 16, the cell of claim 17, or the product made by the method of claim 18 or 19, or the pharmaceutical composition of claim 20; optionally, instructions for use are also included.
CN202280022765.8A 2021-04-01 2022-03-31 Nanometer antibody for resisting Human Serum Albumin (HSA) and application thereof Pending CN117043184A (en)

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