CN117736329A - anti-PD-1 antibodies and uses thereof - Google Patents
anti-PD-1 antibodies and uses thereof Download PDFInfo
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- CN117736329A CN117736329A CN202311797802.5A CN202311797802A CN117736329A CN 117736329 A CN117736329 A CN 117736329A CN 202311797802 A CN202311797802 A CN 202311797802A CN 117736329 A CN117736329 A CN 117736329A
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Abstract
The present disclosure provides an antibody or antigen binding fragment thereof that specifically binds PD-1. The method obtains the Pembrolizumab mutant with greatly improved affinity by carrying out mutation screening on the CDR region of the Pembrolizumab, can be better used for improving the drug effect of the Pembrolizumab, expands the double-antibody design molecular form of the Pembrolizumab, and enhances the targeting of the Pembrolizumab and cytokine fusion protein drugs.
Description
Reference to sequence Listing
The present application comprises a sequence listing in computer readable form, which is incorporated herein by reference.
Technical Field
The present disclosure relates to the field of antibodies, and more particularly to an antibody to PD-1 and uses thereof.
Background
Programmed death factor 1 (PD-1) is a CD28 family member that is expressed primarily on the surface of cd4+ T cells, cd8+ T cells, NKT cells, B cells and activated monocytes. PD-1 is expressed by induction of T Cell Receptor (TCR) or B Cell Receptor (BCR) signaling (Immunol Rev,2010, 236:219-242). PD-1 is an immunosuppressive receptor (Nat Rev Drug Discov,2013,12 (2): 130-146). PD-1 has two ligands: PD-L1 and PD-L2. The interaction of PD-1 between adjacent cells and its ligand PD-L1 or PD-L2 can inhibit the transmission of TCR signal channel, the effects of cell proliferation, transcriptional activation and cytokine production mediated by TCR, etc., and effectively inhibit the immune response of immune T cells. Tumor cells can escape immune surveillance in vivo by over-expressing PD-L1 (International Immunology,2007,19 (7): 813-824 2007). By blocking the interaction of PD1 and PD-L1, the immunosuppression of tumor cells PD-L1 on T cells is relieved, and the tumor cell killing activity of CD8+ T cells can be remarkably improved.
PD-L1 expression is detected on the surfaces of various tumor cells such as lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, renal cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, head and neck cancer and the like. Studies have shown that high expression of PD-L1 on tumor cells is highly correlated with clinically poor prognosis in cancer patients (International Immunology,2007,19 (7): 813-824 2007).
There are a variety of PD-1 antibodies currently on the market, representative of which include the fully human IgG4 (S228P) antibody nivolumab (Cancer ImmunolRes (2014) 1 (9): 846) and the humanized IgG4 (S228P) antibody pembrolizumab (WO 2008156712 and Hamid et al, N Engl J Med (2013) 369:2). Nevertheless, in some cases, there remains a need to provide antibodies that bind human PD-1 with higher affinity than certain prior art antibodies, and there remains a need to provide antibodies that more effectively block interactions of human PD-1 with PD-L1 and PD-L2 than certain prior art antibodies. Higher affinity and better blocking may alone or together translate into greater in vivo activity or lower amounts of desired dosing.
Pembrolizumab (trade name Keystuda, abbreviated as K medicine) is an antitumor drug developed and produced by the company moesadong, belongs to one of PD-1 inhibitors, is the most successful cancer immunotherapy drug at present, and also creates the fastest approval record of Chinese import antitumor biological preparations. However, on the one hand, the current Pembrolizumab has a lower affinity than other antibodies, and there is still a need for improvement. Because the affinity of the Pembrolizumab is low, the effects of the double antibody (1:1) based on the Pembrolizumab, the antibody fusion cytokine and the like are not ideal. Therefore, the affinity of the Pembrolizumab and the PD-1 is improved, and the method has great significance for improving the drug effect of the Pembrolizumab, expanding the double-antibody design molecular form of the Pembrolizumab, enhancing the targeting of the Pembrolizumab and cytokine fusion protein drugs and the like.
Disclosure of Invention
In response to the above-described problems, the present disclosure provides antibodies, methods of making the same, compositions, and the like. The benefits provided by the present disclosure are broadly applicable to the fields of antibody therapy and diagnostics, and can be used in conjunction with antibodies that react with a variety of targets.
The invention discloses an isolated antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof specifically binds PD-1 and comprises a heavy chain variable region (VH) and a light chain variable region (VL),
the heavy chain variable region comprises:
HCDR1 comprising a sequence corresponding to SEQ ID NO:7 or a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity to SEQ ID NO:7, forming;
(ii) HCDR2 comprising an amino acid sequence corresponding to SEQ ID NO:8 and 9 or a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity or consisting of SEQ ID NO:8 and 9; and
(iii) HCDR3 comprising an amino acid sequence corresponding to SEQ ID NO:10 or a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity or consisting of SEQ ID NO:10, the composition is as follows;
the light chain variable region comprises:
(i) LCDR1 comprising a sequence corresponding to SEQ ID NO:11-14 or a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity or consisting of SEQ ID NO:11-14 parts; and
(ii) LCDR2 comprising a sequence corresponding to SEQ ID NO:15 or a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity or consisting of SEQ ID NO: 15; and
(iii) LCDR3 comprising a sequence corresponding to SEQ ID NO:16 or a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity or consisting of SEQ ID NO: 16;
wherein said LCDR1 is relative to SEQ ID NO:11 has the following substitutions: Y11I, L, V, wherein according to SEQ ID NO: 11.
In some embodiments of the invention, the antibody or fragment, wherein HCDR2 is substantially identical to SEQ ID NO:8 has the following substitutions: S5Y, wherein according to SEQ ID NO: 8.
In some embodiments of the invention, the antibody or fragment comprises a sequence corresponding to SEQ ID NO:2 has a substitution selected from the group consisting of: S54Y.
In some embodiments of the invention, the antibodies or fragments thereof include the following combinations:
(i) SEQ ID NO:7, HCDR1, SEQ ID NO:8, and HCDR2 as set forth in SEQ ID NO:10, HCDR3 shown in SEQ ID NO:12, LCDR1, SEQ ID NO:15, and LCDR2 as set forth in SEQ ID NO: LCDR3 as shown at 16; or (b)
(ii) SEQ ID NO:7, HCDR1, SEQ ID NO:8, and HCDR2 as set forth in SEQ ID NO:10, HCDR3 shown in SEQ ID NO:13, LCDR1, SEQ ID NO:15, and LCDR2 as set forth in SEQ ID NO: LCDR3 as shown at 16; or (b)
(iii) SEQ ID NO:7, HCDR1, SEQ ID NO:8, and HCDR2 as set forth in SEQ ID NO:10, HCDR3 shown in SEQ ID NO:14, LCDR1, SEQ ID NO:15, and LCDR2 as set forth in SEQ ID NO: LCDR3 as shown at 16; or (b)
(iv) SEQ ID NO:7, HCDR1, SEQ ID NO:9, and HCDR2 as set forth in SEQ ID NO:10, HCDR3 shown in SEQ ID NO:13, LCDR1, SEQ ID NO:15, and LCDR2 as set forth in SEQ ID NO: LCDR3 as shown at 16; or (b)
(v) SEQ ID NO:7, HCDR1, SEQ ID NO:9, and HCDR2 as set forth in SEQ ID NO:10, HCDR3 shown in SEQ ID NO:14, LCDR1, SEQ ID NO:15, and LCDR2 as set forth in SEQ ID NO: LCDR3 as shown at 16; or (b)
(vi) SEQ ID NO:7, HCDR1, SEQ ID NO:9, and HCDR2 as set forth in SEQ ID NO:10, HCDR3 shown in SEQ ID NO:12, LCDR1, SEQ ID NO:15, and LCDR2 as set forth in SEQ ID NO:16, LCDR3.
In some embodiments of the invention, the antibody or fragment comprises one of the following combinations:
(i) A heavy chain comprising a sequence identical to SEQ ID NO:1 or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:1, the composition is as follows;
a light chain comprising a sequence identical to SEQ ID NO:3 or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:3, composing;
(ii) A heavy chain comprising a sequence identical to SEQ ID NO:1 or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:1, the composition is as follows;
a light chain comprising a sequence identical to SEQ ID NO:4 or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:4, the composition is formed;
(iii) A heavy chain comprising a sequence identical to SEQ ID NO:1 or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:1, the composition is as follows;
a light chain comprising a sequence identical to SEQ ID NO:5 or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:5, composing;
(iv) A heavy chain comprising a sequence identical to SEQ ID NO:6, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:6, composition;
a light chain comprising a sequence identical to SEQ ID NO:4 or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:4, the composition is formed;
(v) A heavy chain comprising a sequence identical to SEQ ID NO:6, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:6, composition;
a light chain comprising a sequence identical to SEQ ID NO:5 or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:5 composition
(vi) A heavy chain comprising a sequence identical to SEQ ID NO:6, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:6, composition;
a light chain comprising a sequence identical to SEQ ID NO:3 or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 3.
In some embodiments of the invention, the antibody or fragment, wherein the antibody is selected from the group consisting of: whole antibodies, bispecific antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.
In some embodiments of the invention, the antibody or fragment, wherein the fragment is selected from the group consisting of: fab fragments, fab' fragments, F (ab) 2 Fragments, fv fragments and ScFv.
In another aspect, the invention provides an isolated nucleic acid molecule comprising a nucleic acid sequence encoding said antibody or fragment.
In another aspect, the invention provides a vector comprising said nucleic acid molecule.
In another aspect, the invention provides a host cell comprising said nucleic acid molecule or said vector.
In another aspect, the invention provides a conjugate comprising said antibody or fragment coupled to at least one detectable label.
In another aspect, the invention also provides an antibody drug conjugate comprising an antibody comprising one or more drug moieties covalently linked to the antibody or fragment directly or via a linker.
In another aspect, the invention provides a multispecific molecule comprising an antibody or antigen-binding fragment of the invention; preferably, the multispecific molecule specifically binds PD-1, and additionally specifically binds one or more other targets; further preferred, the multispecific molecule further comprises at least one molecule having a second binding specificity for a second target.
In another aspect, the invention provides a pharmaceutical composition or kit comprising said antibody or fragment, or said nucleic acid molecule, or said vector, or said host cell, or said conjugate, or said antibody drug conjugate, or said multispecific molecule, and a pharmaceutically acceptable carrier.
In another aspect, the invention also provides the use of said antibody or fragment, or said nucleic acid molecule, or said vector, or said host cell, or said conjugate, or said antibody drug conjugate, or said multispecific molecule, or said pharmaceutical composition or kit, in the preparation of a kit for diagnosing, detecting or monitoring a disease associated with PD-1 expression.
In another aspect, the invention also provides the use of said antibody or fragment, or said nucleic acid molecule, or said vector, or said host cell, or said conjugate, or said antibody drug conjugate, or said multispecific molecule, or said pharmaceutical composition or kit, in the manufacture of a medicament for treating a disease associated with PD-1 expression or determining the prognosis thereof.
In another aspect, the disease associated with PD-1 expression is a cancer selected from the group consisting of: lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, renal cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer.
According to the invention, through mutation screening of the CDR region of Pembrolizumab, the Pembrolizumab mutant with greatly improved affinity (for example, 11 times of Pembrolizumab) can be better used for improving the drug effect of Pembrolizumab, expanding the double-antibody design molecular form of Pembrolizumab and enhancing the targeting of Pembrolizumab and cytokines.
Drawings
The accompanying drawings are included to provide a further understanding of the disclosure, and are incorporated in and constitute a part of this specification, illustrate embodiments of the disclosure and together with the description serve to explain the disclosure, without limitation to the disclosure.
FIGS. 1A-1E are graphs showing the results of detection of the binding activity of Pembrolizumab and its mutant antibodies to the membrane surface PD-1 of the Jurkat-hPD-1 stably transformed cell line, wherein the final concentration of the antibodies is taken as the abscissa and the units are nM, and the fluorescence intensity signal of the cells is detected as the ordinate;
FIGS. 2A-2E are results of biological activity assays of Pembrolizumab and its mutant antibodies, wherein Log10 post-value is taken as the abscissa for final antibody concentration, nM for antibody concentration, and the detected signal value of RLU is taken as the ordinate, and RLU represents the relative luminescence unit.
Detailed Description
In order that those skilled in the art will better understand the present invention, a clear and complete description of the technical solutions of the examples of the present invention will be provided below, it being apparent that the examples described are merely illustrative of some, but not all, embodiments of the present invention. Therefore, the invention is not limited to the specific embodiments illustrated. Furthermore, any section headings used herein are not to be construed as limiting the subject matter described.
Unless otherwise defined herein, scientific and technical terms used in connection with the present invention will have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context requires otherwise, terms in the singular shall include the plural and terms in the plural shall include the singular. More specifically, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. In this application, the use of "or" means "and/or" unless stated otherwise. Furthermore, the use of the term "include" and other forms (such as "include" and "contain") is not limiting. Furthermore, the scope provided in the specification and the appended claims includes all values between the endpoints and between the endpoints.
Definition of the definition
For a better understanding of the present invention, definitions and explanations of related terms are provided below.
The term "antibody" or "Ab" generally refers to a Y-shaped tetrameric protein comprising two heavy (H) and two light (L) polypeptide chains held together by covalent disulfide bonds and non-covalent interactions. The light chain of an antibody can be classified as either a kappa or lambda light chain. Heavy chains can be classified as mu, delta, gamma, alpha or epsilon, which define the isotype of the antibody as IgM, igD, igG, igA or IgE, respectively. In the light and heavy chains, the variable region is linked to the constant region by a "J" region of about 12 or more amino acids, and the heavy chain also comprises a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of 3 domains (CH 1, CH2 and CH 3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions can be further divided into hypervariable regions (called complementarity determining regions, CDRs, for short) separated by relatively conserved regions (called framework regions, FR, for short). Each VH and VL consists of 3 CDRs and 4 FR in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 from the N-terminal to the C-terminal. CDRs on VH are HCDR1, HCDR2 and HCDR3; the CDRs on VL are LCDR1, LCDR2, LCDR3. The variable regions (VH and VL) of each heavy/light chain pair form antigen binding sites/portions, respectively. The distribution of amino acids in various regions or domains follows the numbering definitions in common systems such as Kabat, IMGT or Chothia, and in particular embodiments of the present disclosure, the determination of CDR sequences uses the numbering definitions in Kabat systems.
Antibodies in the present disclosure also include antigen-binding portions (interchangeably used with the term "antigen-binding fragment"). Antigen binding portionRefers to a polypeptide comprising a fragment of an intact antibody that retains the ability to specifically bind to an antigen to which the full length or intact antibody specifically binds, and/or that competes for binding to the same antigen as the full length antibody. Under some conditions, the antigen binding portion includes Fab, fab ', F (ab') 2 Fd, fv, dAb and Complementarity Determining Region (CDR) fragments, single chain antibodies (e.g., scFv), chimeric antibodies, diabodies and antibodies comprising at least a portion sufficient to confer specific antigen binding capacity to a polypeptide. The antigen binding portion of an antibody can be obtained from a given antibody by conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods), and the specificity can be screened in the same manner as for an intact antibody.
The term "isotype" refers to the class of antibodies (e.g., igM or IgG 1) encoded by the heavy chain constant region gene.
The term "monoclonal antibody" or "mAb" refers to an antibody molecule/preparation consisting of single molecules. Monoclonal antibodies exhibit a single binding specificity and affinity for a particular epitope. Antibodies of the invention may be derived from different species including, but not limited to, mice, rats, rabbits, guinea pigs, and humans.
The term "epitope" refers to an antigenic determinant in a molecule, and refers to a portion of a molecule that is recognized by the immune system (e.g., by an antibody), such as a discrete three-dimensional site on an antigen that is recognized by the immune system. In the present invention, the epitope shown is, for example, PD-1 protein.
The term "chimeric antibody" as used herein refers to antibodies whose variable region sequences are from one species and constant region sequences are from another species, e.g., antibodies in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody.
The term "humanized antibody" is intended to refer to an antibody in which CDR sequences/antigen binding portions or sites derived from the germline of another mammalian species, such as a mouse, have been grafted onto a human framework sequence. In addition, additional framework region modifications can be made within the human framework sequence.
The term "KD value" is the equilibrium dissociation constant between an antibody and its antigen, i.e., the koff/kon or KD/ka (as determined by SPR techniques) ratio. Thus the lower the KD value (lower concentration), the higher the affinity of the antibody. Thus "KD values" can be used to measure the binding affinity of an antibody to its antigen.
The terms "PD-1" and "PD-1 antigen" are used interchangeably herein to include any variant, isoform, and species homolog of human PD-1 that is naturally expressed by a cell or that is expressed on a cell transfected with a PD-1 gene. In some embodiments, binding of an antibody of the disclosure to a PD-1 antigen mediates killing of cells expressing PD-1 (e.g., tumor cells) by inactivating PD-1. Killing of PD-1 expressing cells may occur through one or more of the following mechanisms: cell death/apoptosis induction, ADCC and CDC.
The term "anti-PD-1 antibody" or "PD-1 antibody" refers to an antibody as defined herein that is capable of binding to a PD-1 antigen or to a cell expressing PD-1.
The term "specific binding" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen against which it is directed.
The term "isolated" refers to a state obtained from a natural state by manual means. If a certain "isolated" substance or component occurs naturally, it may be due to a change in its natural environment, or the substance is separated from the natural environment, or both. For example, a polynucleotide or polypeptide that is not isolated naturally occurs in a living animal, and the same polynucleotide or polypeptide in high purity isolated from that natural state is referred to as an isolated polynucleotide or polypeptide. The term "isolated" does not exclude mixed artificial or synthetic substances nor other impure substances that do not affect the activity of the isolated substances. For example, the isolated antibody may be substantially free of other cellular material and/or chemicals.
The term "vector" refers to a nucleic acid vector into which a polynucleotide may be inserted. When a vector allows expression of a protein encoded by a polynucleotide inserted therein, the vector is referred to as an expression vector. The vector may be transformed, transduced or transfected into a host cell to express the carried genetic material element in the host cell. Vectors are well known to those of skill in the art and include, but are not limited to, plasmids, phages, cosmids, artificial chromosomes such as Yeast Artificial Chromosomes (YACs), bacterial Artificial Chromosomes (BACs) or P1-derived artificial chromosomes (PACs); phages such as lambda phage or M13 phage and animal viruses. Animal viruses that may be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, papovaviruses (e.g., SV 40). The vector may contain a number of elements for controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may comprise an origin of replication. For the vector expressing the antibody, a vector type in which the heavy chain and the light chain of the antibody are present in different vectors or a vector type in which the heavy chain and the light chain are present in the same vector may be used.
The term "host cell" refers to a cellular system that can be engineered to produce a protein, protein fragment, or peptide of interest. Host cells include, but are not limited to, cultured cells, e.g., mammalian cultured cells derived from rodents (rat, mouse, guinea pig, or hamster), such as CHO, BHK, NSO, SP2/0, YB2/0; or human tissue or hybridoma cells, yeast cells, and insect cells, and cells contained within a transgenic animal or cultured tissue. The term encompasses not only the particular subject cell, but also the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not be identical to the parent cell, but are still included within the scope of the term "host cell".
The term "identity" refers to the relationship between the sequences of two or more polypeptide molecules (or protein molecules) or two or more nucleic acid molecules as determined by aligning and comparing the sequences. "percent identity" refers to the percentage of identical residues between amino acids or nucleotides in a comparison molecule and is calculated based on the size of the smallest molecule being compared. For these calculations, the gaps in the alignment (if any) are preferably addressed by a specific mathematical model or computer program (i.e., an "algorithm"). Methods that can be used to calculate identity of aligned nucleic acids or polypeptides include those described in Computational Molecular Biology, (Lesk, a.m., ed.), 1988,New York:Oxford University Press; biocomputingInformatics and Genome Projects, (Smith, d.w., ed.), 1993,New York:AcademicPress; computer Analysis of Sequence Data Part I, (Griffin, a.m., and Griffin, h.g., eds.), 1994,New Jersey:Humana Press; von Heinje, g.,1987,Sequence Analysisin Molecular Biology,New York:Academic Press; sequence Analysis Primer, (Gribskov, m.and deveeux, j., eds.), 1991,New York:M.Stockton Press; and carilo et al,1988,SIAMJ.Applied Math.48:1073.
The term "immunogenicity" refers to the ability to stimulate the formation of specific antibodies or sensitized lymphocytes in an organism. It refers not only to the nature of antigens to stimulate the activation, proliferation and differentiation of specific immune cells to ultimately produce immune effector substances such as antibodies and sensitized lymphocytes, but also to the fact that specific immune responses of antibodies or sensitized T lymphocytes can develop in the immune system of an organism after stimulation of the organism with an antigen. Immunogenicity is the most important property of an antigen. Whether an antigen is able to successfully induce the generation of an immune response in a host depends on three factors: the nature of the antigen, the reactivity of the host and the means of immunization.
The term "transfection" refers to the process of introducing nucleic acid into eukaryotic cells, particularly mammalian cells. Protocols and techniques for transfection include, but are not limited to, lipofection and chemical and physical methods such as electroporation. Numerous transfection techniques are well known in the art and are disclosed herein. See, e.g., graham et al, 1973,Virology 52:456; sambrook et al, 2001,Molecular Cloning:A Laboratory Manual, supra; davis et al, 1986,Basic Methods in Molecular Biology,Elsevier; chu et al,1981, gene 13:197.
The term "hybridoma" and the term "hybridoma cell line" are used interchangeably. When referring to the term "hybridoma" and the term "hybridoma cell line", they also include subclones and progeny cells of the hybridoma.
The term "immune effector function" includes any function mediated by a component of the immune system that results in inhibition of tumor growth and/or inhibition of tumorigenesis, as well as inhibition of tumor dissemination and metastasis. Preferably, immune effector function results in killing tumor cells. Preferably, the immune effector function in the present invention is an antibody-mediated effector function. Such functions include Complement Dependent Cytotoxicity (CDC), antibody dependent cell mediated cytotoxicity (ADCC), induction of apoptosis in cells bearing tumor-associated antigens (e.g., by binding of antibodies to surface antigens), and/or inhibition of proliferation of cells bearing tumor-associated antigens, preferably ADCC and/or CDC. Antibodies can also function simply by binding to tumor-associated antigens on the surface of tumor cells. For example, an antibody may block the function of a tumor-associated antigen or induce apoptosis simply by binding to a tumor-associated antigen on the surface of a tumor cell.
The term "cancer" refers to solid and non-solid tumors such as leukemia mediated by growth, proliferation or metastasis of any tumor or malignant cell that is responsible for a medical condition. For example, cancers associated with PD-1 expression or caused by abnormal expression thereof, including but not limited to: b-cell lymphomas, including NHL, pre-B-cell lymphocytic leukemia/lymphoma and mature B-cell neoplasms, such as B-cell Chronic Lymphocytic Leukemia (CLL)/Small Lymphocytic Lymphoma (SLL), B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, mantle Cell Lymphoma (MCL), follicular Lymphoma (FL), including low-, medium-and high-grade FL, cutaneous follicular central lymphoma, marginal zone B-cell lymphoma (MALT type, intranode and spleen type), hairy cell leukemia, diffuse large B-cell lymphoma, burkitt's lymphoma, plasma cell myeloma, post-transplant lymphoproliferative disorder, fahrenheit macroglobulinemia and Anaplastic Large Cell Lymphoma (ALCL)
The term "pharmaceutically acceptable" means that the carrier, diluent, excipient and/or salt thereof is chemically and/or physically compatible with the other ingredients of the formulation, and physiologically compatible with the recipient.
The term "pharmaceutically acceptable carrier and/or excipient" refers to a carrier that is pharmacologically and/or physiologically compatible with the subject and the active agentBodies and/or excipients, which are well known in the art (see, e.g., remington's Pharmaceutical sciences. Mediated by Gennaro AR,19 th Pennsylvania: mack Publishing Company, 1995) and includes, but is not limited to, pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, pH modifiers include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, such as Tween-80; ionic strength enhancers include, but are not limited to, sodium chloride.
The term "adjuvant" refers to a non-specific immunopotentiator that, when delivered to an organism with an antigen or delivered to an organism in advance, can enhance the immune response to an antigen or alter the type of immune response in an organism. There are various adjuvants including, but not limited to, aluminum adjuvants (e.g., aluminum hydroxide), freund's adjuvants (e.g., freund's complete adjuvant and Freund's incomplete adjuvant), corynebacterium parvum, lipopolysaccharide, cytokines, and the like. Freund's adjuvant is the most commonly used adjuvant in current animal experiments. Aluminum hydroxide adjuvants are more commonly used in clinical trials.
anti-PD-1 antibodies
In some aspects, the invention includes an isolated antibody or antigen-binding fragment thereof.
In the context of the present application, "antibody" may include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized and primate antibodies, CDR-grafted antibodies, human antibodies, recombinantly produced antibodies, intracellular antibodies, bifunctional antibodies, multispecific antibodies, bispecific antibodies, monovalent antibodies, multivalent antibodies, anti-idiotypic antibodies, synthetic antibodies, including muteins and variants thereof, modified antibodies; and derivatives thereof (including Fc fusion proteins and other modifications), as well as any other immunoreactive molecules, so long as they exhibit preferential association or binding with PD-1 proteins. Furthermore, unless the context dictates otherwise, the term also includes all classes of antibodies (i.e., igA, igD, igE, igG and IgM) and all subclasses (i.e., igG1, igG2, igG3, igG4, igA1, and IgA 2). In a preferred embodiment, the antibody is a monoclonal antibody. In a more preferred embodiment, the antibody is a chimeric monoclonal antibody or a humanized monoclonal antibody or a modified chimeric monoclonal antibody.
The variable regions and CDRs in an antibody sequence can be identified according to the general rules already developed in the art (as described above, e.g., kabat) numbering system or by aligning the sequences with a database of known variable regions.
In some embodiments, an isolated antibody or antigen-binding fragment thereof may comprise a conservative substitution or modification or substitution (e.g., a conservative substitution), a deletion or addition (e.g., a substitution, a deletion or addition of 1, 2, 3, 4, or 5 amino acids; or a conservative substitution of up to 20, up to 15, up to 10, or up to 5 amino acids), of an amino acid in the variable region of the heavy and/or light chain, or has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100% sequence identity to the antibody or antigen fragment thereof from which it is derived. Amino acid deletion variants comprising deletions at the N-and/or C-terminus of the protein are also referred to as N-and/or C-terminal truncation variants (truncation variant). It is understood in the art that certain modifications of conserved sequences that do not eliminate antigen binding may be made. See, for example, brummel et al (1993) Biochem 32:1180-8; de Wildt et al (1997) Prot.Eng.10:835-41; komissarov et al (1997) J.biol. Chem.272:26864-26870; hall et al (1992) J.Immunol.149:1605-12; kelley and O' Connell (1993) biochem.32:6862-35; adib-Conquy et al (1998) int. Immunol.10:341-6and beer et al (2000) Clin. Can. Res.6:2835-43.
As described above, the term "conservative substitution/conservative substitution" as used herein refers to an amino acid substitution/substitution that does not adversely affect or alter the basic properties of a protein/polypeptide comprising an amino acid sequence. For example, conservative substitutions/conservative substitutions may be introduced by standard techniques known in the art (e.g., site-directed mutagenesis and PCR-mediated mutagenesis). Conservative amino acid substitutions include substitutions in which an amino acid residue is substituted by another amino acid residue having a similar side chain, such as a substitution of a physically or functionally similar residue (e.g., of similar size, shape, charge, chemical nature including the ability to form covalent or hydrogen bonds, etc.) to the corresponding amino acid residue. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, the corresponding amino acid residue is preferably substituted with another amino acid residue from the same side chain family. Methods for identifying amino acid conservative substitutions are well known in the art (see, e.g., brummell et al, biochem.32:1180-1187 (1993); kobayashi et al, protein Eng.12 (10): 879-884 (1999); and Burks et al, proc. Natl. Acad. Sci. USA94:412-417 (1997), which is incorporated herein by reference).
For example Y34I, L, V, Y representing position 34 of the amino acid sequence may be substituted with any one of I, L and V.
Regardless of how the antibody is produced, methods of testing the ability of the antibody to bind to an antigen (e.g., PD-1) are known in the art and include any antibody-antigen binding assay, such as Radioimmunoassays (RIA), ELISA, western blots, immunoprecipitation, SPR, and competitive inhibition assays (see, e.g., janeway et al, below and U.S. patent application publication No. 2002/0197266, and the above section on competition assays).
According to the invention, in standard assays (e.g., the assays described in the present invention), if an antibody has significant affinity to a predetermined target (e.g., PD-1 protein or PD-1 expressing cells), the antibody is able to bind to the predetermined target, flow Cytometry (FCM) may be used in order to test the binding of monoclonal antibodies to living cells expressing PD-1. Preferably, in flow cytometric fluorescence sorting (FACS) analysis, the binding of the antibody to a target expressed on the cell surface is determined, and the antibody is capable of binding to the target with an "affinity" if it binds detectably to the target (PD-1 protein or PD-1 expressing cells).
The PD-1 specificity according to the invention means that it is capable of binding to one or more PD-1 epitopes, in particular PD-1 epitopes in their native conformation, in particular human PD-1 specificity.
The engineering of desired properties of an antibody without altering the properties is varied in the art by various means, such as the manner in which the light and heavy chains of an antibody are recombined, amino acid substitutions made, etc., as employed in the present disclosure. For example, sequences of the invention, including chimeric or humanized antibody sequences, may be subjected to conservative amino acid substitutions.
Antibodies interact with target antigens primarily through amino acid residues located in the Complementarity Determining Regions (CDRs) of the six heavy and light chains. For this reason, the amino acid sequences of CDRs are more diverse between antibodies than other sequences. Since CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of a particular naturally occurring antibody by constructing expression vectors that contain CDR sequences from the particular naturally occurring antibody that are grafted onto framework sequences from different antibodies with different properties (see, e.g., riechmann, L. Et al (1998) Nature 332:323-327; jones, P. Et al (1986) Nature321:522-525; and Queen, C. Et al (1989) Proc. Natl. Acad. Sci. U.S.A.86:10029-10033). Such framework sequences may be obtained from public DNA databases including germline antibody gene sequences. These lines are sequences that differ from the mature antibody gene sequence in that they do not contain the fully assembled variable genes that are formed by V (D) J ligation during B cell maturation. Germline gene sequences will also have sequences that differ from the high affinity second antibody repertoire (secondary repertoire antibody) at individual points evenly across the variable region.
Mouse antibodies are highly immunogenic in humans, resulting in reduced therapeutic efficacy when repeatedly applied, with major immunogenicity mediated by the heavy chain constant region. If the individual antibodies are chimeric or humanized, the immunogenicity of the mouse antibodies in humans can be reduced or completely avoided.
Chimeric antibodies refer to antibodies in which different portions are derived from different animal species, e.g., antibodies having variable regions derived from mouse antibodies and human immunoglobulin constant regions. The variable regions of the heavy and light chains of a mouse antibody are joined to the constant regions of the human heavy and light chains to obtain chimerism of the antibody (e.g., as described in Kraus et al, in Methods in MolecularBiology series, recombinant antibodies for cancer therapy ISBN-0-89603-918-8). In a preferred embodiment, the chimeric antibody is produced by ligating a human kappa light chain constant region to a mouse light chain variable region. In another preferred embodiment, chimeric antibodies can be produced by linking a human lambda light chain constant region to a mouse light chain variable region.
By humanized antibody is meant an antibody in which CDR sequences/antigen binding portions or sites derived from the germline of another mammalian species, such as a mouse, are grafted onto a human framework sequence.
To reduce immunogenicity of antibodies to humans, humanized anti-PD-1 antibodies are produced using the sequences of the PD-1 antibodies of the present disclosure, utilizing the CDR regions of murine anti-PD-1 antibodies in combination with framework regions of human origin (e.g., human immunoglobulins) to form humanized anti-PD-1 antibodies of the present disclosure, the humanized antibodies are expected to retain the function of binding to human PD-1 as well as the function of binding to monkey PD-1.
Preparation or production of antibodies
Antibodies of the invention can be produced by a variety of techniques, including conventional monoclonal antibody methods, such as kohlerand milstein, nature256:495 The standard somatic hybridization technique of (1975), and other techniques for producing monoclonal antibodies, such as viral or oncogene transformed B lymphocytes or phage display techniques using antibody gene libraries, somatic hybridization, and in turn, for example, by genetic engineering recombinant techniques, may be employed. For example, DNA molecules encoding the heavy and light chain genes of the antibodies of the invention are obtained by chemical synthesis or PCR amplification, the resulting DNA molecules are inserted into an expression vector, then the host cells are transfected, then the transfected host cells are cultured under specific conditions, and the antibodies of the invention are expressed.
Other preferred animal systems for the preparation of monoclonal antibody secreting hybridomas are the rat and rabbit systems (e.g., described in Spieker-Poletetal, proc. Natl. Acad. Sci. U.S. A.92:9348 (1995), see also Rossietal. Am. J. Clin. Pathol.124:295 (2005)). Hybridoma production in mice is a very well established method. Immunization protocols and techniques for isolating immunized splenocytes for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion methods are also known.
Monoclonal antibodies may be prepared using a variety of techniques known in the art, including hybridoma techniques, recombinant techniques, phage display techniques, transgenic animals, or some combination thereof. For example, hybridomas and art-recognized biochemical and genetic engineering techniques can be used to produce monoclonal antibodies, as described in detail in An, zhiqiang (ed.) Therapeutic Monoclonal Antibodies: from Bench to Clinic, johnWiley and Sons,1st ed.2009; shire et al (eds.) Current Trends in MonoclonalAntibody Development and Manufacturing, spring science+ Business Media LLC,1 step.2010; harlow et al Antibodies A Laboratory Manual, cold Spring HarborLaboratory Press,2nd ed 1988; hammerling, et al, in Monoclonal Antibodies and T-Cell hybrid 563-681 (Elsevier, N.Y., 1981), each of which is incorporated herein by reference in its entirety.
It will be appreciated that the selected binding sequences may be further altered, e.g., to increase affinity for the target, to humanize the target binding sequence, to improve its production in cell culture, to reduce its immunogenicity in vivo, to produce multispecific antibodies, and the like, and antibodies comprising altered target binding sequences are also antibodies of the invention.
In some embodiments, the method of producing an antibody or fragment described in the present disclosure comprises the steps of:
(i) Expressing the antibody or fragment in a host cell; and optionally
(ii) Isolating the antibody or antigen binding fragment thereof from the host cell.
In a preferred embodiment, the anti-PD-1 monoclonal antibody is prepared by using a hybridoma.
To obtain hybridomas producing antibodies of the invention, e.g., human monoclonal antibodies of the invention, spleen cells and/or lymph node cells from immunized mice can be isolated and fused to a suitable immortalized cell line, e.g., a mouse myeloma cell line. The hybridomas produced are screened for the production of antigen-specific antibodies. The generation of hybridomas is well known in the art. See, e.g., harlow and Lane (1988) Antibodies, A Laboratory Manual, cold SpringHarbor Publications, new York.
Antibodies of the invention may also be produced in host cell transfectomas using, for example, a combination of recombinant DNA techniques and gene transfection methods well known in the art (e.g., morrison, s. (1985) Science 229:1202). In some embodiments, DNA encoding part or full length light and heavy chains obtained by standard molecular biology techniques is inserted into one or more expression vectors, such that the genes are operably linked to transcriptional and translational regulatory sequences. In this context, the term "operably linked" is intended to mean that the antibody genes are linked into a vector such that transcriptional and translational control sequences within the vector perform their intended functions of regulating the transcription and translation of the antibody genes.
The antibody light chain gene and the antibody heavy chain gene may be inserted into the same or different expression vectors. In some embodiments, the variable region is used to generate a full length antibody gene of any antibody isotype by inserting it into an expression vector that already encodes the heavy and light chain constant regions of the desired isotype, such that the VH segment is operably linked to the CH segment and the VL segment within the vector, and the CL segment within the vector. Additionally or alternatively, the recombinant expression vector may encode a signal peptide that facilitates secretion of the antibody chain from the host cell. The antibody chain gene may be cloned into a vector such that the signal peptide is linked to the amino terminus of the antibody chain gene. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).
To express the light and heavy chains, expression vectors encoding the heavy and light chains are transfected into host cells by standard techniques. The various forms of the term "transfection" are intended to encompass the various techniques commonly used for introducing exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like. Antibodies of the invention can be expressed in prokaryotic or eukaryotic host cells, such as mammalian host cells, which can assemble and secrete properly folded and immunocompetent antibodies.
Mammalian host cells for expression of recombinant antibodies of the invention include chinese hamster ovary cells (CHO cells) (including DHFR CHO cells described in Urlaub and Chasin, (1980) proc.Natl.Acad.Sci.USA 77:4216-4220), NSO myeloma cells, COS cells and SP2 cells for use with a DHFR selection marker (e.g., as described in R.J. Kaufman and P.A. Sharp (1982) J.MoI.biol.159:601-621). In particular, for use with NSO myeloma, another expression system is the GS gene expression system disclosed in WO 87/04462, WO 89/01036 and EP 338,841. When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a period of time sufficient to allow expression of the antibody in the host cell or by secreting the antibody into the medium in which the host cell is grown. Antibodies can be recovered from the culture medium using standard protein purification methods.
In another preferred embodiment, transgenic or transchromosomal mice with a partially human immune system (rather than a mouse system) may be used to generate human monoclonal antibodies against PD-1.
Another strategy for generating monoclonal antibodies is to isolate the genes encoding the antibodies directly from the antibody-producing lymphocytes of the defined strategy, see for example babcock et al, 1996; anovel strategy for generating monoclonal antibodies from single, isolated lymphocytes producing antibodies of defined strategy. Details of recombinant antibody engineering are also found in WelschofandKrau, recombinant antibodes for cancer therapy ISBN-0-89603-918-8and Benny K.C.Lo Antibody Engineering ISBN1-58829-092-1.
To prepare chimeric antibodies, the murine immunoglobulin variable region can be linked to a human immunoglobulin constant region using methods known in the art (see, e.g., U.S. Pat. No. 4,816,567 to Capilli et al). An isolated nucleic acid encoding a VH region can be converted to a full length heavy chain gene by operably linking the nucleic acid encoding the VH region to another DNA molecule encoding a heavy chain constant region (CH 1, CH2, and CH 3). The sequence of a human heavy chain constant region gene is known in the art (see, e.g., kabat et al (1991), sequences Of Proteins of Immunological Interest, fifth Edition, U.S. device of Health and Human Services, NIH Publication No. 91-3242). The heavy chain constant region may be an IgG1, igG2, igG3, igG4, igA, igE, igM or IgD constant region, but more preferably is an IgG1 or IgG4 constant region. The isolated nucleic acid encoding the VL region can be converted to a full length light chain gene (as well as a Fab light chain gene) by operably linking the DNA encoding the VL to another DNA molecule encoding the light chain constant region CL. The sequences of human light chain constant region genes are known in the art (see, e.g., kabat et al, supra), and DNA fragments comprising these regions can be obtained by standard PCR amplification. In preferred embodiments, the light chain constant region may be a kappa or lambda constant region, but is generally preferred. Once the DNA fragments encoding the VH and VL segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, such as converting the variable region genes into full-length antibody chain genes, fab fragment genes or scFv genes. In these operations, a DNA fragment encoding a VL or VH is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or flexible linker. The term "operably linked" as used herein is intended to mean that the two DNA fragments are linked such that the amino acid sequences encoded by the two DNA fragments remain in frame.
To prepare humanized antibodies, murine CDR regions can be inserted into a human framework sequence using methods known in the art (see U.S. Pat. No. 5,225,539 to Winter; U.S. Pat. No. 5,530,101 to Queen et al; U.S. Pat. No. 5,585,089; U.S. Pat. No. 5,693,762; and Lo, benny, K.C., editor, in Antibody Engineering: methods and Protocols, volume 248,Humana Press,New Jersey,2004). Alternatively, transgenic animals can also be utilized that are capable of producing no endogenous immunoglobulins upon immunization and are capable of producing a fully human antibody repertoire. For example, homozygous deletion of the antibody heavy chain Junction (JH) gene in chimeric and germ-line mutant mice has been reported to completely suppress endogenous antibody production, and then transferring an array of human germ-line immunoglobulin genes into the germ-line mutant mice will result in the mice producing human antibodies upon encountering antigen stimulation (see, e.g., jakobovits et al, 1993,Proc.Natl.Acad.Sci.USA 90:2551;Jakobovits et al, 1993, nature362:255-258; bruggermann et al, 1993,Year in Immunology7:33; and Duchosal et al, 1992,Nature 355:258). Non-limiting examples of such transgenic animals include, huMAb mice (Medarex, inc.) containing human immunoglobulin gene miniloci (miniloci) encoding unrearranged human heavy (μ and γ) and kappa light chain immunoglobulin sequences, together with targeted mutations that inactivate endogenous μ and kappa chain loci (see, e.g., lonberg et al (1994) Nature 368 (6474): 856-859); or "KM mouse TM" carrying both human heavy chain transgenes and human light chain transchromosomes (see patent application WO 02/43478). Other methods of antibody humanization include phage display techniques (Hoogenboom et al, 1991, J. Mol. Biol.227:381; marks et al, J. Mol. Biol.1991, 222:581-597; vaughan et al, 1996,Nature Biotech 14:309).
Nucleic acid molecules encoding antibodies of the invention
In some aspects, the invention relates to isolated nucleic acid molecules comprising a nucleic acid sequence encoding an isolated antibody or fragment thereof as described above in the present disclosure.
The nucleic acids of the invention may be obtained using standard molecular biology techniques. For hybridoma-expressed antibodies (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes as described further below), cdnas encoding the light and heavy chains of antibodies prepared by the hybridomas can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from immunoglobulin gene libraries (e.g., using phage display techniques), nucleic acids encoding such antibodies can be recovered from the gene library.
To prepare chimeric antibodies, the murine immunoglobulin variable region can be linked to a human immunoglobulin constant region using methods known in the art (see, e.g., U.S. Pat. No.4,816,567 to Capilli et al). By operably linking a nucleic acid encoding a VH to another DNA molecule encoding heavy chain constant regions (CH 1, CH2 and CH 3), the isolated nucleic acid encoding the VH region can be converted to a full length heavy chain gene, and DNA fragments comprising these regions can be obtained by standard PCR amplification. The isolated nucleic acid encoding the VL region can be converted to a full length light chain gene (as well as a Fab light chain gene) by operably linking the DNA encoding the VL to another DNA molecule encoding the light chain constant region CL. Once the DNA fragments encoding the VH and VL segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, such as converting the variable region genes into full-length antibody chain genes, fab fragment genes or scFv genes. In these operations, a DNA fragment encoding a VL or VH is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or flexible linker.
Conjugate(s)
In one aspect, the present disclosure provides a conjugate comprising an antibody or fragment thereof as described above coupled to at least one detectable label. Detectable labels include, but are not limited to: (i) providing a detectable signal; (ii) Interact with a second label to modify a detectable signal provided by the first or second label, such as FRET (fluorescence resonance energy transfer ); (iii) The mobility (e.g., electrophoretic mobility) is affected by charge, hydrophobicity, shape, or other physical parameters, or (iv) a capture moiety is provided, such as affinity, antibody/antigen, or ion complexation.
Suitable structures for the label are, for example, fluorescent labels, luminescent labels, chromophore labels, radioisotope labels, isotopic labels, preferably stable isotopic labels, isobaric labels (isobaric label), enzymatic labels (e.g. horseradish peroxidase, alkaline phosphatase, beta-galactosidase, urease, glucose oxidase, etc.), particle labels (especially metal particle labels, magnetic particle labels, polymeric particle labels), organic small molecules (e.g. biotin, ligands or binding molecules of receptors (e.g. cell adhesion proteins or lecithins), label sequences comprising nucleic acid and/or amino acid residues detectable by use of binding agents, etc. labels include, but are not limited to, barium sulfate, ioxitic acid, iodic acid, calcium amiodarone, sodium diatrizoate, diatrizonamine, meglumine, sodium caseinate and radiodiagnostic agents (including emitters, (e.g. fluoro-18 and carbon-11), gamma emitters (e.g. iodo-123, iodo-99, iodo-co-99, gadolinium-111), fluorescent substances (e.g. fluoro-co-111), fluorescent substances (e.g. g. fluorescent substances) and fluorescent substances (e.g. g. gold-co-gold) and latex).
The detectable labels described above can be detected by methods known in the art. For example, fluorescent markers may be detected using a photodetector to detect the emitted light. Enzyme labels are typically detected by providing a substrate to an enzyme and detecting a reaction product produced by the action of the enzyme on the substrate. In certain embodiments, such labels can be suitable for immunological detection (e.g., enzyme-linked immunoassay, radioimmunoassay, fluorescent immunoassay, chemiluminescent immunoassay, etc.). In certain embodiments, a detectable label as described above may be attached to an antibody or antigen binding fragment thereof of the invention by linkers of different lengths to reduce potential steric hindrance.
Antibody drug conjugates/immunoconjugates
In one aspect, the present disclosure provides an antibody drug conjugate comprising an antibody comprising one or more drug moieties/therapeutic agents linked (e.g., covalently linked) directly or via a linker to an antibody or fragment thereof as described previously. In the antibody-drug conjugate of the present application, the linker structure for conjugating the anti-PD-1 antibody to the drug is not particularly limited as long as the resulting antibody-drug conjugate can be used.
Because of the ability of the antibody-drug conjugates to selectively deliver one or more drugs to a target tissue (e.g., a tumor-associated antigen, such as a tumor expressing PD-1), the antibody-drug conjugates can increase the therapeutic efficacy of the antibodies or antigen-binding fragments thereof of the invention in treating a disease (e.g., cancer).
Multispecific molecules
The antibodies or antigen binding fragments thereof of the invention can be used to form multispecific molecules (e.g., bispecific molecules). The antibodies or antigen binding fragments thereof of the invention may be part of a multispecific molecule (e.g., a bispecific molecule) that comprises a second functional moiety (e.g., a second antibody) or a third functional moiety (e.g., a third antibody) having a binding specificity different from the antibodies or antigen binding fragments thereof of the invention, thereby being capable of binding to at least two different binding sites and/or target molecules. For example, an antibody or antigen-binding fragment thereof of the invention may be linked to a second antibody or antigen-binding fragment thereof capable of specifically binding any protein that may be used as a potential target for combination therapy. To produce the bispecific or multispecific molecules, an antibody or antigen-binding fragment thereof of the invention may be linked (e.g., by chemical coupling, gene fusion, non-covalent association, or other means) to one or more other binding molecules (e.g., additional antibodies, antibody fragments, peptides, or binding mimics).
Thus, in some aspects, the invention provides a multispecific molecule comprising an antibody, or antigen-binding fragment thereof, of the invention.
In certain preferred embodiments, the multispecific molecule specifically binds PD-1 (e.g., human PD-1 or monkey PD-1) and specifically binds one or more other targets.
In certain preferred embodiments, the multispecific molecule further comprises at least one molecule (e.g., a second antibody) having a second binding specificity for a second target.
In certain preferred embodiments, the multispecific molecule is a bispecific antibody.
Pharmaceutical composition
In some aspects, the present disclosure provides a pharmaceutical composition or kit comprising an antibody or fragment as described above, a nucleic acid molecule as described above, a vector as described above, a host cell as described above, a conjugate as described above, an antibody drug conjugate as described above, a multispecific molecule as described above; and a pharmaceutically acceptable carrier.
The pharmaceutical composition may optionally contain one or more additional pharmaceutically active ingredients, such as another antibody or drug. The pharmaceutical compositions of the invention may also be administered in combination with, for example, another immunostimulant, anticancer agent, antiviral agent, or vaccine, such that the anti-PD-1 antibodies enhance the immune response to the vaccine. Pharmaceutically acceptable carriers can include, for example, pharmaceutically acceptable liquid, gel or solid carriers, aqueous media, nonaqueous media, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending/dispersing agents, chelating agents, diluents, adjuvants, excipients or non-toxic auxiliary substances, combinations of various components known in the art or more.
Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavouring agents, thickening agents, colouring agents, emulsifying agents or stabilizing agents such as sugars and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, thioglycolic acid, thiosorbitol, butylmethylanisole, butylated hydroxytoluene and/or propyl arsenate. As disclosed herein, the antibodies or antigen-binding fragments thereof of the disclosed compositions may be oxidized in a solvent containing one or more antioxidants, such as methionine, that reduce the antibodies or antigen-binding fragments thereof. Redox can prevent or reduce the decrease in binding affinity, thereby enhancing antibody stability and extending shelf life. Thus, in some embodiments, the invention provides compositions comprising one or more antibodies or antigen binding fragments thereof and one or more antioxidants, such as methionine. The invention further provides methods wherein the antibody or antigen-binding fragment thereof is admixed with one or more antioxidants, such as methionine, such that the antibody or antigen-binding fragment thereof may be protected from oxidation to extend its shelf life and/or increase activity.
For further illustration, pharmaceutically acceptable carriers may include, for example, aqueous vehicles such as sodium chloride injection, ringer's injection, isotonic dextrose injection, sterile water injection, or dextrose and lactate ringer's injection, non-aqueous vehicles such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil or peanut oil, antimicrobial agents of bacteriostatic or fungistatic concentration, isotonic agents such as sodium chloride or glucose, buffers such as phosphate or citrate buffers, antioxidants such as sodium bisulfate, local anesthetics such as procaine hydrochloride, suspending and dispersing agents such as sodium carboxymethyl cellulose, hydroxypropyl methylcellulose or polyvinylpyrrolidone, emulsifying agents such as polysorbate 80 (TWEEN-80), sequestering or chelating agents such as EDTA (ethylenediamine tetraacetic acid) or EGTA (ethylene glycol tetraacetic acid), ethanol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid or lactic acid. The antimicrobial agent used as a carrier may be added to a pharmaceutical composition in a multi-dose container containing phenols or cresols, mercuric preparations, benzyl alcohol, chlorobutanol, methyl and propyl parahydroxybenzoates, thimerosal, benzalkonium chloride and benzethonium chloride. Suitable excipients may include, for example, water, saline, dextrose, glycerol, or ethanol. Suitable non-toxic auxiliary substances may include, for example, wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrins.
Administration, formulation and dosage
The pharmaceutical compositions of the invention may be administered to a subject in need thereof in vivo by a variety of routes including, but not limited to, oral, intravenous, intra-arterial, subcutaneous, parenteral, intranasal, intramuscular, intracranial, intracardiac, intraventricular, intratracheal, buccal, rectal, intraperitoneal, intradermal, topical, transdermal and intrathecal, or by implantation or inhalation. The compositions of the present invention may be formulated as solid, semi-solid, liquid or gaseous forms of formulation; including but not limited to tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalants and aerosols. The appropriate formulation and route of administration may be selected depending upon the intended application and treatment regimen.
Suitable formulations for enteral administration include hard or soft gelatin capsules, pills, tablets (including coated tablets), elixirs, suspensions, syrups or inhalants and controlled release dosage forms thereof.
Formulations suitable for parenteral administration (e.g., by injection) include aqueous or nonaqueous, isotonic, pyrogen-free, sterile liquids (e.g., solutions, suspensions) in which the active ingredient is dissolved, suspended, or otherwise provided (e.g., in liposomes or other microparticles). These liquids may additionally contain other pharmaceutically acceptable ingredients such as antioxidants, buffers, preservatives, stabilizers, bacteriostats, suspending agents, thickening agents and solutes which render the formulation isotonic with the blood (or other relevant body fluids) of the intended recipient. Examples of excipients include, for example, water, alcohols, polyols, glycerol, vegetable oils, and the like. Examples of isotonic carriers suitable for use in such formulations include sodium chloride injection, ringer's solution or lactated ringer's injection. Similarly, the particular dosage regimen (including dosage, time and repetition) will depend on the particular individual and medical history of the individual, and empirical considerations such as pharmacokinetics (e.g., half-life, clearance rate, etc.).
The requirements for effective pharmaceutical carriers for injectable formulations/compositions are well known to those of ordinary skill in the art (see, e.g., pharmaceutics and Pharmacy Practice, J.B. Lippincott Company, philadelphia, pa., banker and Chalmers editions, pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, toissel, 4 th edition, pages 622-630 (1986)).
The frequency of administration can be determined and adjusted during treatment and based on reducing the number of proliferating or tumorigenic cells, maintaining such a reduction in tumor cells, reducing proliferation of tumor cells or delaying the development of metastasis. In some embodiments, the administered dose may be adjusted or reduced to control potential side effects and/or toxicity. Alternatively, sustained continuous release formulations of the therapeutic compositions of the present invention may be suitable.
Those skilled in the art will appreciate that the appropriate dosage may vary from patient to patient. Determining the optimal dose generally involves balancing the level of therapeutic benefit with any risk or adverse side effects. The dosage level selected will depend on a variety of factors including, but not limited to, the activity of the particular compound, the administration, the time of administration, the rate of clearance of the compound, the duration of treatment, other co-administered drugs, compounds and/or materials, the severity of the condition, as well as the species, sex, age, weight, condition, general health and previous medical history of the patient. The dosage is typically selected to achieve a local concentration at the site of action that achieves the desired effect without causing substantial deleterious or adverse side effects.
In general, the antibodies or antigen-binding fragments thereof of the invention may be administered in a variety of ranges.
In certain preferred embodiments, the course of treatment involving an antibody or antigen-binding fragment thereof of the invention will comprise multiple doses of the selected pharmaceutical product administered over a period of weeks or months. More specifically, the antibodies or antigen binding fragments thereof of the invention may be administered daily, every two days, every four days, weekly, every ten days, every two weeks, every three weeks, monthly, every six weeks, every two months, every ten weeks, or every three months. In this regard, it is understood that the dosage or adjustment interval may be varied based on patient response and clinical practice.
A compatible formulation for parenteral administration (e.g., intravenous injection) will comprise an antibody or antigen-binding fragment thereof as disclosed herein at a concentration of about 5 μg/mL to about 100 mg/mL.
The antibodies of the invention may be co-administered with one or more other therapeutic agents (e.g., cytotoxic agents, radiopharmaceuticals, anti-neoplastic agents, anti-angiogenic agents, or and immunosuppressants) to reduce induction of an immune response against the antibodies of the invention. The antibody may be linked to the therapeutic agent (as an immune complex) or may be administered separately from the therapeutic agent.
In the context of administration of a treatment, the term "combination" or "co-administration" as used herein refers to the use of more than one treatment or therapeutic agent. The use of the term "combination" does not limit the order of treatments or therapeutic agents administered to a subject. The treatment or therapeutic agent may be administered prior to, concurrently with, or after the administration of the second treatment or therapeutic agent to the patient. Preferably, the therapeutic or therapeutic agents are administered to the subject in a sequence, amount, and/or over a time interval such that the therapeutic or therapeutic agents may act together. In a particular embodiment, the therapeutic or therapeutic agents are administered to the subject in a sequence, amount, and/or over a time interval such that they provide increased benefits over if administered otherwise (particularly independently of each other). Preferably, the added benefit is a synergistic effect.
Medical application
The antibodies, antibody compositions and methods of the invention have a number of in vitro and in vivo uses, including, for example, detection of PD-1 or enhancement of immune responses. For example, these molecules may be administered to cultured cells in vitro or ex vivo, or to human subjects in vivo, for example.
Preferred subjects include mammals, such as humans/patients. Mammals in the context of the present invention are humans, non-human primates, domesticated animals such as dogs, cats, sheep, cattle, goats, pigs, horses, etc., laboratory animals such as mice, rats, rabbits, guinea pigs, etc., and farm animals such as zoo animals.
Treatment of disorders associated with PD-1 expression
In some aspects, the invention provides methods of treating a disorder in a mammal comprising administering to a subject (e.g., human) in need of treatment a therapeutically effective amount of an antibody or antigen-binding fragment thereof disclosed herein.
As described herein, the antibodies of the present disclosure have one or more activities that can therapeutically apply killer cells and/or inhibit cells. In particular, killing cells, inhibiting proliferation of cells, and/or inhibiting colony formation of cells can be used to treat or prevent cancer (including cancer metastasis). Inhibition of cell proliferation, colony formation and/or metastasis may be employed, inter alia, for the treatment or prevention of cancer metastasis and metastatic spread of cancer cells.
In some aspects, the disclosure provides a method for treating or determining prognosis of a disease associated with PD-1 expression in a subject, comprising administering to a subject in need thereof an effective dose of the antibody or antigen-binding fragment thereof, the nucleic acid molecule, the vector, the host cell, the conjugate, the antibody drug conjugate, the multispecific molecule, or the pharmaceutical composition or kit.
In some aspects, the disclosure provides the antibody or antigen-binding fragment thereof, the nucleic acid molecule, the vector, the host cell, the conjugate, the antibody drug conjugate, the multispecific molecule, or the pharmaceutical composition or kit for use in a method of treating a disease associated with PD-1 expression or determining the prognosis thereof in a subject.
In some aspects, the disclosure provides the use of the antibody or antigen binding fragment thereof, the nucleic acid molecule, the vector, the host cell, the conjugate, the antibody drug conjugate, the multispecific molecule, or the pharmaceutical composition or kit in the preparation of a reagent (or drug) for treating a disease associated with PD-1 expression or determining the prognosis thereof.
In one embodiment, the disease associated with PD-1 expression includes a neoplastic disease, such as cancer.
The antibody or antigen binding fragment thereof may be used alone as a monotherapy or may be used in combination with chemotherapy or radiation therapy.
The antibody or antigen binding fragment thereof may be used in combination with an anticancer agent, a cytotoxic agent, or a chemotherapeutic agent.
The term "anti-cancer agent" or "antiproliferative agent" means any agent that can be used to treat cell proliferative disorders such as cancer, and includes, but is not limited to, cytotoxic agents, cytostatic agents, anti-angiogenic agents, radiation therapy and radiation therapeutic agents, targeted anti-cancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormonal therapy, radiation therapy, anti-metastatic agents and immunotherapeutic agents. It will be appreciated that in selected embodiments as described above, such anti-cancer agents may comprise conjugates and may be conjugated to the disclosed site-specific antibodies prior to administration. More specifically, in certain embodiments, a selected anti-cancer agent is linked to a unpaired cysteine of an engineered antibody to provide an engineered conjugate as described herein. Thus, such engineered conjugates are expressly contemplated as being within the scope of the present invention. In other embodiments, the disclosed anti-cancer agents will be administered in combination with site-specific conjugates comprising different therapeutic agents as described above.
Diagnosis of
The present invention provides in vitro and in vivo methods for detecting, diagnosing or monitoring proliferative disorders and methods of screening cells from a patient to identify tumor cells, including tumorigenic cells. Such methods comprise identifying an individual having cancer for treatment or monitoring progression of cancer, comprising contacting a patient or a sample obtained from the patient (in vivo or in vitro) with an antibody described herein, and detecting the presence or absence or level of binding of the bound antibody to a bound or free target molecule in the sample. In some embodiments, the antibody will comprise a detectable label or a reported molecule as described herein.
In some aspects, the disclosure provides a method of diagnosing, detecting, or monitoring a disease associated with PD-1 expression, comprising administering to a subject in need thereof an effective dose of the antibody or antigen-binding fragment thereof, the nucleic acid molecule, the vector, the host cell, the conjugate, the antibody drug conjugate, the multispecific molecule, or the pharmaceutical composition or kit.
In some aspects, the disclosure provides the antibody or antigen-binding fragment thereof, the nucleic acid molecule, the vector, the host cell, the conjugate, the antibody drug conjugate, the multispecific molecule, or the pharmaceutical composition or kit for use in a method of diagnosing, detecting, or monitoring a disease associated with PD-1 expression in a subject.
In yet another aspect, the disclosure provides the use of the antibody or antigen binding fragment thereof, the nucleic acid molecule, the vector, the host cell, the conjugate, the antibody drug conjugate, the multispecific molecule, or the pharmaceutical composition or kit in the preparation of a reagent (or drug) for diagnosing, detecting, or monitoring a disease associated with PD-1 expression.
Samples may be analyzed by a variety of assays, such as radioimmunoassays, enzyme immunoassays (e.g., ELISA), competitive binding assays, fluorescent immunoassays, immunoblot assays, western blot analysis, and flow cytometry assays. Compatible in vivo diagnostic or diagnostic assays may include imaging or monitoring techniques known in the art, such as magnetic resonance imaging, computerized tomography (e.g., CAT scan), positron emission tomography (e.g., PET scan), radiography, ultrasound, and the like, as known to those skilled in the art.
The methods described herein for detecting or monitoring PD-1 expression or the level of cells expressing PD-1 in vitro can also be used for non-diagnostic purposes.
Preferred subjects include mammals, such as humans/patients in need thereof.
The sample from the subject is blood, fecal matter (urine or faeces), oral or nasal secretions, or alveolar lavage, interstitial fluid, sweat or extracts thereof from the subject.
Pharmaceutical package and kit
Pharmaceutical packages and kits comprising one or more containers of one or more doses of the antibodies or antigen binding fragments thereof are also provided. In certain embodiments, unit doses are provided, wherein the unit doses contain a predetermined amount of a composition comprising, for example, an antibody or antigen-binding fragment thereof, with or without one or more other agents. For other embodiments, such unit doses are supplied in single use, pre-filled syringes. In other embodiments, the compositions contained in the unit dose may comprise saline, sucrose, or the like; buffers such as phosphates and the like; and/or formulated in a stable and effective pH range. Alternatively, in certain embodiments, the conjugate composition may be provided as a lyophilized powder, which may be reconstituted upon addition of a suitable liquid (e.g., sterile water or saline solution). In certain preferred embodiments, the composition comprises one or more substances that inhibit protein aggregation, including, but not limited to, sucrose and arginine. Any label on or associated with the container indicates that the encapsulated conjugate composition is to be used to treat the selected neoplastic disease condition.
Such kits typically comprise a pharmaceutically acceptable formulation of the engineered conjugate in a suitable container, and optionally one or more anticancer agents or other agents in the same or different containers. The kit may also contain other pharmaceutically acceptable formulations for diagnostic or combination therapy.
More specifically, the kits may have a single container containing the antibodies or antigen binding fragments thereof of the disclosure, with or without additional components, or they may have different containers for each desired agent. Where a combination therapeutic agent is provided for conjugation, a single solution may be pre-mixed in molar equivalent combination or with more of one component than the other. Alternatively, the conjugate of the kit and any optional anticancer agent may be stored separately in separate containers prior to administration to the patient. The kit may further comprise a second/third container means for holding a sterile pharmaceutically acceptable buffer or other diluent, such as bacteriostatic water for injection (BWFI), phosphate Buffered Saline (PBS), ringer's solution and dextrose solution.
When the components of the kit are provided in one or more liquid solutions, the liquid solution is preferably an aqueous solution, particularly preferably a sterile aqueous solution or a saline solution. However, the components of the kit may be provided as a dry powder. When the reagents or components are provided in dry powder form, the powder may be reconstituted by the addition of a suitable solvent. It is contemplated that the solvent may also be provided in another container.
Examples
The invention generally described herein will be understood more readily by reference to the following examples, which are provided by way of illustration and are not intended to limit the invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The raw materials, reagent materials and the like used in the examples described below are commercially available products unless otherwise specified.
Hereinafter, "Pembrolizumab and its mutant antibody" are also collectively referred to as "PD-1 antibody".
Example 1 preparation of Pembrolizumab and its mutant antibodies
And (3) respectively cloning the light chain codon and the heavy chain codon of the Pembrolizumab and the mutant antibody sequence thereof to an expression vector pCDNA3.4 (Sieimer-Fed), co-transfecting the ExpiCHO-S cells (Sieimer-Fed) for expression, collecting the supernatant, and purifying by Protein A (Cytiva) to obtain candidate antibody proteins. Pembrolizumab and its mutant antibody sequences are shown in Table 1.
TABLE 1Pembrolizumab and mutant antibody sequences thereof
Example 2 affinity detection of Pembrolizumab and its mutant antibodies with human PD-1
Using Biacore 8K, capture antibody was performed using Protein A chip (Cytiva), pembrolizumab and its mutant antibody were diluted to about 1. Mu.g/ml, and about 100RU was captured (RU was response unit or response unit, i.e., response value. In literature relating to surface plasmon resonance technology, it was shown that if Protein-Protein interaction analysis was performed using CM5 chip, 1RU approximately corresponds to 1pg/mm generated on the chip surface 2 Concentration variation. Biacore systemThe response value of (c) should always be in RU instead of in concentration. ) The method comprises the steps of carrying out a first treatment on the surface of the Binding hPD-his (Acro, PD 1-H5221), concentrations 100, 50, 25, 12.5, 6.25, 0nM, binding 80s, dissociation 120s, regeneration Glycine1.5 (Cytiva) 30s,30ul/min.
The results are shown in table 2, where the affinity of the Pembrolizumab mutant antibodies was improved compared to Pembrolizumab, with the LC34L mutant having the highest affinity, 11.3 times that of Pembrolizumab.
TABLE 2Pembrolizumab and detection of affinity of mutant antibodies to human PD-1 antigen
Example 3 binding of Pembrolizumab and its mutant antibodies to Jurkat-hPD-1 cell membrane surface PD-1 binding of the PD-1 antibody to Jurkat-hPD-1 cells (Gibby organism) engineered by single point mutation was detected using a flow-through method. The specific operation is as follows: 2E+05 Jurkat-hPD-1 cells were incubated with PD-1 antibody (25 nM initial concentration, 4-fold gradient dilution, 8 concentration points) at 2-8deg.C for 1h in the absence of light. Washing for 2 times, adding goat anti-human IgG Fc specific-FITC antibody (sigma-Aldrich) diluted according to the ratio of 1:100, incubating for 30min at 2-8 ℃ in the dark, adding staining buffer to resuspend cells after washing for 3 times, detecting the fluorescence intensity of the cells by using a flow meter (BD FACS Lyric), taking the value after Log10 as the abscissa of the final concentration of the antibody, carrying out nonlinear fitting by taking the median value of the detected fluorescence intensity signal of the cells as the ordinate, and calculating the EC50 value. FIGS. 1A-1E are data showing the binding of a portion of a single point mutant PD-1 antibody to Jurkat-hPD-1 cell membrane surface PD-1. The results show that: the binding activity of Pembrolizumab and the mutant antibody thereof with the PD-1 on the surface of the Jurkat-hPD-1 cell membrane is improved, wherein the signal peak value of the binding of the LC34L, HC Y-LC34I, HC Y-LC34V mutant antibody with the PD-1 on the surface of the Jurkat-hPD-1 cell membrane is obviously improved.
Example 4 biological Activity detection of Pembrolizumab and its mutant antibodies
The biological activity of the PD-1 antibody is detected by the reporter gene method, and the assay consists of two genetically engineered cell lines: PD-1 EffectCells (h_pd-1 Reporter Jurkat Cell Line), i.e., jurkat T cells stably expressing the human PD-1 receptor and transcription factor-induced luciferase reporter; PD-L1 aAPC/CHO-K1 cells (aAPC (OKT 3) PDL1CHO-K1 Cell Line) are CHO-K1 cells stably expressing human PD-L1 and a Cell surface protein that activates the cognate TCR in an antigen-independent manner. All 2 cells were derived from the gemini organism. When both cells are co-cultured, the interaction between PD-1/PD-L1 inhibits TCR signaling and transcription factor mediated expression of luciferases. Upon addition of an antibody blocking PD-1/PD-L1, this inhibition is released, causing TCR signaling and transcription factor mediated expression of luciferases. The specific operation is as follows: inoculating 2.5E+04 aAPC (OKT 3) PDL1CHO-K1 Cell Line cells into a white 96-well Cell culture plate, and placing at 37deg.C and 5% CO 2 Incubating for 16-20H, removing aAPC (OKT 3) PDL1CHO-K1 Cell Line supernatant after incubation, adding H_PD-1Reporter Jurkat Cell Line,50 μl/well adjusted to a density of 2E 6/ml; PD-1 antibody (initial 450nM, 3-fold gradient dilution, 8 concentration spots) was added, 50. Mu.l/well; at 37℃with 5% CO 2 Incubate under conditions for 6h. Bright-Lite Luciferase Assay System (Vazyme, DD 1204-03) was added, 100. Mu.l/well; incubation is carried out at room temperature for 5-10min, and luminescence signal values are detected by a multifunctional enzyme-labeled instrument (MD, spectraMax i 3X). And taking the value of the final concentration of the antibody after Log10 as an abscissa, and taking the detected LUM signal value as an ordinate to perform nonlinear fitting, so as to calculate an IC50 value. FIGS. 2A-2E are partial single point mutant PD-1 antibody biological activity data. The results show that: the biological activity of the LC34V, LC34I, LC34L, HC54Y-LC34I, HC Y-LC34V mutant antibody is obviously enhanced.
Incorporated by reference
The entire contents of each of the patent documents and scientific documents mentioned herein are incorporated by reference for all purposes.
Equivalency of
The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The above embodiments should therefore be regarded as illustrative in all respects, rather than limiting on the invention described herein. The scope of the invention is, therefore, indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are intended to be embraced therein.
Claims (9)
1. An isolated antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof specifically binds to PD-1 and comprises a heavy chain variable region (VH) and a light chain variable region (VL),
the heavy chain variable region comprises:
(i) HCDR1 comprising a sequence corresponding to SEQ ID NO:7 or a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity to SEQ ID NO:7, forming;
(ii) HCDR2 comprising an amino acid sequence corresponding to SEQ ID NO:8 and 9 or a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity or consisting of SEQ ID NO:8 and 9; and
(iii) HCDR3 comprising an amino acid sequence corresponding to SEQ ID NO:10 or a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity or consisting of SEQ ID NO:10, the composition is as follows;
the light chain variable region comprises:
(i) LCDR1 comprising a sequence corresponding to SEQ ID NO:11, a sequence having at least 80%, at least 85%, at least 95% sequence identity; and
(ii) LCDR2 comprising a sequence corresponding to SEQ ID NO:15 or a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity or consisting of SEQ ID NO: 15; and
(iii) LCDR3 comprising a sequence corresponding to SEQ ID NO:16 or a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity or consisting of SEQ ID NO: 16;
Wherein said LCDR1 is relative to SEQ ID NO:11 has the following substitutions: Y11I, L, V, wherein according to SEQ ID NO: 11.
2. The antibody or fragment of claim 1, wherein HCDR2 is compared to SEQ ID NO:8 has the following substitutions: S5Y, wherein according to SEQ ID NO: 8.
3. The antibody or fragment of any one of claims 1 or 2, comprising a combination of:
(i) SEQ ID NO:7, HCDR1, SEQ ID NO:8, and HCDR2 as set forth in SEQ ID NO:10, HCDR3 shown in SEQ ID NO:12, LCDR1, SEQ ID NO:15, and LCDR2 as set forth in SEQ ID NO: LCDR3 as shown at 16; or (b)
(ii) SEQ ID NO:7, HCDR1, SEQ ID NO:8, and HCDR2 as set forth in SEQ ID NO:10, HCDR3 shown in SEQ ID NO:13, LCDR1, SEQ ID NO:15, and LCDR2 as set forth in SEQ ID NO: LCDR3 as shown at 16; or (b)
(iii) SEQ ID NO:7, HCDR1, SEQ ID NO:8, and HCDR2 as set forth in SEQ ID NO:10, HCDR3 shown in SEQ ID NO:14, LCDR1, SEQ ID NO:15, and LCDR2 as set forth in SEQ ID NO: LCDR3 as shown at 16; or (b)
(iv) SEQ ID NO:7, HCDR1, SEQ ID NO:9, and HCDR2 as set forth in SEQ ID NO:10, HCDR3 shown in SEQ ID NO:13, LCDR1, SEQ ID NO:15, and LCDR2 as set forth in SEQ ID NO: LCDR3 as shown at 16; or (b)
(v) SEQ ID NO:7, HCDR1, SEQ ID NO:9, and HCDR2 as set forth in SEQ ID NO:10, HCDR3 shown in SEQ ID NO:14, LCDR1, SEQ ID NO:15, and LCDR2 as set forth in SEQ ID NO: LCDR3 as shown at 16; or (b)
(vi) SEQ ID NO:7, HCDR1, SEQ ID NO:9, and HCDR2 as set forth in SEQ ID NO:10, HCDR3 shown in SEQ ID NO:12, LCDR1, SEQ ID NO:15, and LCDR2 as set forth in SEQ ID NO:16, LCDR3.
4. The antibody or fragment of any one of claims 1-3, comprising one of the following combinations:
(i) A heavy chain comprising a sequence identical to SEQ ID NO:1 or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:1, the composition is as follows;
a light chain comprising a sequence identical to SEQ ID NO:3 or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:3, composing;
(ii) A heavy chain comprising a sequence identical to SEQ ID NO:1 or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:1, the composition is as follows;
a light chain comprising a sequence identical to SEQ ID NO:4 or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:4, the composition is formed;
(iii) A heavy chain comprising a sequence identical to SEQ ID NO:1 or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:1, the composition is as follows;
a light chain comprising a sequence identical to SEQ ID NO:5 or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:5, composing;
(iv) A heavy chain comprising a sequence identical to SEQ ID NO:6, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:6, composition;
a light chain comprising a sequence identical to SEQ ID NO:4 or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:4, the composition is formed;
(v) A heavy chain comprising a sequence identical to SEQ ID NO:6, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:6, composition;
a light chain comprising a sequence identical to SEQ ID NO:5 or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:5, composing;
(vi) A heavy chain comprising a sequence identical to SEQ ID NO:6, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:6, composition;
a light chain comprising a sequence identical to SEQ ID NO:3 or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 3.
5. The antibody or fragment of any one of the above claims, wherein the antibody is selected from the group consisting of: whole antibodies, bispecific antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.
6. The antibody or fragment of any one of the preceding claims, wherein the fragment is selected from the group consisting of: fab fragments, fab' fragments, F (ab) 2 Fragments, fv fragments and ScFv.
7. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding an antibody or fragment according to any one of claims 1-6.
8. A multispecific molecule comprising the antibody or antigen-binding fragment of any one of claims 1-6; preferably, the multispecific molecule specifically binds to a PD-1 antibody and additionally specifically binds to one or more other targets; further preferred, the multispecific molecule further comprises at least one molecule having a second binding specificity for a second target.
9. Use of an antibody or fragment according to any one of claims 1 to 6, or a nucleic acid molecule according to claim 7, or a multispecific molecule according to claim 8 in the manufacture of a medicament for treating a disease associated with PD-1 antibody expression or determining the prognosis thereof.
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Citations (3)
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CN110022907A (en) * | 2016-10-10 | 2019-07-16 | 国家生物技术研究所公司 | Modified cells of non-cell toxicity and application thereof |
WO2020014419A2 (en) * | 2018-07-11 | 2020-01-16 | Momenta Pharmaceuticals Inc | Compositions and methods related to engineered fc-antigen binding domain constructs targeted to pd-l1 |
CN113195537A (en) * | 2018-10-22 | 2021-07-30 | 融合抗体公开有限公司 | Antibody libraries and methods |
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CN110022907A (en) * | 2016-10-10 | 2019-07-16 | 国家生物技术研究所公司 | Modified cells of non-cell toxicity and application thereof |
WO2020014419A2 (en) * | 2018-07-11 | 2020-01-16 | Momenta Pharmaceuticals Inc | Compositions and methods related to engineered fc-antigen binding domain constructs targeted to pd-l1 |
CN113195537A (en) * | 2018-10-22 | 2021-07-30 | 融合抗体公开有限公司 | Antibody libraries and methods |
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