阅读说明：本技术 一种人源化t细胞活化的v域免疫抑制因子抗原结合片段 (Humanized T cell activated V domain immunosuppressive factor antigen binding fragment ) 是由 熊浩 于 2019-11-08 设计创作，主要内容包括：本发明涉及抗体领域,特别地涉及一种人源化T细胞活化的V域免疫抑制因子抗原结合片段,本发明的抗原结合片段,稳定性好,具有较强的T细胞功能调控活性和良好的药代动力学特性,能够在体内显著抑制肿瘤的生长。(The invention relates to the field of antibodies, in particular to a humanized T cell activated V domain immunosuppressive factor antigen binding fragment.)

1. A humanized T cell activated V domain immunosuppressive factor antigen binding fragment, said antigen binding fragment comprising a light chain variable region and a heavy chain variable region; the light chain variable region comprises CDR-L1 with an amino acid sequence shown as SEQ ID NO.1, and/or CDR-L2 with an amino acid sequence shown as SEQ ID NO. 2, and/or CDR-L3 with an amino acid sequence shown as SEQ ID NO. 3; the heavy chain variable region comprises a CDR-H1 with an amino acid sequence shown as SEQ ID NO. 4, and/or a CDR-H2 with an amino acid sequence shown as SEQ ID NO. 5, and/or a CDR-H3 with an amino acid sequence shown as SEQ ID NO. 6, and the antigen binding fragment is an antigen binding fragment which specifically binds to a V domain immunosuppressive factor activated by T cells.

2. The antigen-binding fragment according to claim 1, wherein the amino acid sequence of the heavy chain variable region is a variant sequence having at least 95% identity to any one of the amino acid sequences shown in SEQ ID Nos. 7, 9, 10 and 11 and retaining the corresponding biological activity or a variant sequence obtained by deletion, substitution and/or addition of one or more amino acid residues and retaining the corresponding biological activity.

3. The antigen-binding fragment according to claim 1, wherein the amino acid sequence of the light chain variable region is a variant sequence having at least 95% identity to any one of the amino acid sequences shown in SEQ ID Nos. 8, 12, 13 and 14 and retaining the corresponding biological activity or a variant sequence obtained by deletion, substitution and/or addition of one or more amino acid residues and retaining the corresponding biological activity.

4. The antigen-binding fragment of claim 1, wherein the amino acid sequence of the heavy chain variable region is as shown in any one of SEQ ID nos. 7, 9, 10, and 11 and/or the amino acid sequence of the light chain variable region is as shown in any one of SEQ ID nos. 8, 12, 13, and 14.

5. The antigen-binding fragment of claim 4, wherein the heavy chain variable region has the amino acid sequence shown in SEQ ID No.9 and the light chain variable region has the amino acid sequence shown in SEQ ID No. 12.

6. The antigen-binding fragment of any one of claims 1 to 5, wherein the antigen-binding fragment is selected from the group consisting of Fab, Fab ', F (ab)'2, single chain Fv (scFv), Fv fragments, diabodies, and linear antibodies.

7. The antigen-binding fragment of claim 6, wherein the antigen-binding fragment comprises an amino acid sequence at any Fc-terminus selected from the group consisting of human antibodies IgG1, IgG2, IgG3, IgG 4.

8. The antigen-binding fragment of any one of claims 1 to 7, wherein the antigen-binding fragment is produced by culturing a host cell comprising a nucleotide sequence encoding the antigen-binding fragment of claims 1 to 7 in a culture medium and under suitable culture conditions.

9. The antigen-binding fragment of any one of claims 1 to 8, wherein the antibody, antigen-binding fragment thereof, or variant thereof is conjugated to at least one diagnostic and/or therapeutic agent to form an immunoconjugate;

preferably, the diagnostic agent is selected from one or more of a metal, a fluorescent label, a chemiluminescent label, an ultrasound contrast agent, a photosensitizer;

preferably, the therapeutic agent is selected from one or more of another antigen binding fragment, a cytotoxic agent, a radionuclide, a boron atom, an immunomodulator, an immunoconjugate, an oligonucleotide.

10. Use of humanized T cell activated V domain immunosuppressive factor antigen binding fragments comprising recovering the antibody and antigen binding fragments thereof of claims 1-9 produced from culture medium or from cultured host cells and formulating with pharmaceutically acceptable carrier for the treatment of tumors including but not limited to gastric cancer, pancreatic cancer, gallbladder cancer, liver cancer, colorectal cancer, leukemia, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, uterine sarcoma, prostate cancer, bladder cancer, renal cell carcinoma.

11. The use according to claim 10, said tumor is preferably a pancreatic cancer, ovarian cancer or other VISTA-highly expressed tumor type.

Technical Field

The invention relates to the field of monoclonal antibodies, in particular to a humanized T cell activated V domain immunosuppressive factor antigen binding fragment.

Background

The T cell activated v-domain immune suppressor (VISTA) is a novel inhibitory immune checkpoint protein. The expression of VISTA on tumor cells and its associated regulatory mechanisms are not known. VISTA has been shown to be highly expressed in human ovarian and endometrial cancers. Up-regulation of VISTA in endometrial cancer is associated with VISTA promoter methylation status. VISTA inhibits T cell proliferation and cytokine production in tumor cells and reduces tumor-infiltrating CD8+ T cells in vivo. anti-VISTA antibodies prolong the survival of tumor-bearing mice.

In recent years, Immune Checkpoint Therapy (ICT) has changed the appearance of cancer therapy, but not all tumor types respond well to existing immune checkpoint inhibitors. Clinical studies have shown, however, that the clinical benefit of existing ICT on pancreatic cancer is minimal. Pancreatic cancer treatment methods are various, including surgical operation, chemotherapy, radiotherapy, interventional therapy and the like, but poor curative effect of single treatment method is still crudely described in the back of various treatment methods. Surgery is by far the only possible cure for pancreatic cancer, but many patients have lost the opportunity for radical surgery at the time of diagnosis.

Recently, the university of Texas, MD Anderson cancer center, published a paper "Complex of animal infilterates in melanoma and cultural cancer high hlights VISTA as exogenous target in cultural cancer", at the PNAS journal. The research panel found that VISTA is preferentially expressed at high levels in pancreatic cancer compared to melanoma, and the researchers' studies also provided detailed analysis of primary and metastatic pancreatic cancer immune infiltrates compared to melanoma, which may further help guide immunotherapy strategies for pancreatic cancer treatment.

VISTA is a new immunosuppressive factor in tumor microenvironment and also a new target for tumor immunotherapy, and an effective VISTA antibody medicament is urgently needed clinically.

Disclosure of Invention

In order to solve the above technical problems, the present invention provides a humanized T cell activated V domain immunosuppressive factor antigen binding fragment.

The invention is realized by the following technical scheme:

a humanized T cell activated V domain immunosuppressive factor antigen binding fragment comprising a light chain variable region and a heavy chain variable region; the light chain variable region comprises CDR-L1 with an amino acid sequence shown as SEQ ID NO.1, and/or CDR-L2 with an amino acid sequence shown as SEQ ID NO. 2, and/or CDR-L3 with an amino acid sequence shown as SEQ ID NO. 3; the heavy chain variable region comprises a CDR-H1 with an amino acid sequence shown as SEQ ID NO. 4, and/or a CDR-H2 with an amino acid sequence shown as SEQ ID NO. 5, and/or a CDR-H3 with an amino acid sequence shown as SEQ ID NO. 6, and the antigen binding fragment is an antigen binding fragment which specifically binds to a V domain immunosuppressive factor activated by T cells.

Further, the amino acid sequence of the heavy chain variable region is a variant sequence having at least 95% identity with any one of the amino acid sequences shown in SEQ ID Nos. 7, 9, 10, and 11 and retaining the corresponding biological activity or a variant sequence obtained by deleting, replacing, and/or adding one or more amino acid residues and retaining the corresponding biological activity.

Further, wherein the amino acid sequence of the light chain variable region is a variant sequence having at least 95% identity to any one of the amino acid sequences shown in SEQ ID nos. 8, 12, 13, 14 and retaining the corresponding biological activity or a variant sequence obtained by deletion, substitution and/or addition of one or more amino acid residues and retaining the corresponding biological activity.

Further, the amino acid sequence of the heavy chain variable region is shown in any one of SEQ ID No.7, 9, 10 and 11 and/or the amino acid sequence of the light chain variable region is shown in any one of SEQ ID No.8, 12, 13 and 14.

Furthermore, the amino acid sequence of the heavy chain variable region is shown as SEQ ID No.9, and the amino acid sequence of the light chain variable region is shown as SEQ ID No. 12.

Furthermore, the amino acid sequence of the heavy chain variable region is shown as SEQ ID No.10, and the amino acid sequence of the light chain variable region is shown as SEQ ID No. 13.

Further, the antigen binding fragment is selected from the group consisting of Fab, Fab ', f (ab)'2, single chain Fv (scfv), Fv fragments, diabodies, and linear antibodies.

Further, the antigen binding fragment comprises an amino acid sequence of any Fc-terminus selected from the group consisting of human antibodies IgG1, IgG2, IgG3, IgG 4.

Further, the step of producing the antigen-binding fragment comprises culturing a host cell comprising a nucleotide sequence encoding the antigen-binding fragment in a culture medium and under suitable culture conditions.

Further wherein the antibody, antigen binding fragment thereof or variant thereof is conjugated to at least one diagnostic and/or therapeutic agent to form an immunoconjugate

Preferably, the diagnostic agent is selected from one or more of a metal, a fluorescent label, a chemiluminescent label, an ultrasound contrast agent, a photosensitizer;

preferably, the therapeutic agent is selected from one or more of another antigen binding fragment, a cytotoxic agent, a radionuclide, a boron atom, an immunomodulator, an immunoconjugate, an oligonucleotide.

The invention also provides an application of the humanized T cell activated V domain immunosuppressive factor antigen binding fragment, which comprises the application of the antibody and the antigen binding fragment thereof recovered and produced from a culture medium or cultured host cells and preparing a medicament with a pharmaceutically acceptable carrier for treating tumors, wherein the tumors comprise but are not limited to gastric cancer, pancreatic cancer, gallbladder cancer, liver cancer, colorectal cancer, leukemia, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, uterine sarcoma, prostate cancer, bladder cancer and renal cell carcinoma.

Preferably, the tumor is preferably a pancreatic cancer, ovarian cancer or other VISTA-high expressing tumor type.

The anti-VISTA monoclonal antibody provided by the invention has the advantages of strong specificity, good stability, strong T cell function regulation activity and good pharmacokinetic property, and can significantly inhibit the growth of tumors in vivo. Moreover, the anti-VISTA monoclonal antibody of the invention has strong binding blocking activity on VISTA antigen.

Detailed Description

Definition of

It is well known in the art that an antigen binding domain refers to a region that can specifically interact with a target molecule, such as an antigen, with a high degree of selectivity of action, and that sequences recognizing one target molecule are generally unable to recognize other molecular sequences.

Representative antigen binding domains include: a variable region of an antibody, a structural variant of a variable region of an antibody, a binding domain of a receptor, a ligand binding domain, or an enzyme binding domain.

The binding specificity and avidity of an antibody are determined primarily by the CDR sequences, and variants with similar biological activity can be obtained by readily altering the amino acid sequence of the non-CDR regions according to well-established and well-known techniques of the art.

"variable" refers to the fact that certain segments of the variable domains differ significantly in sequence between antibodies, the hypervariable regions being 3 to 12 amino acids in length each, primarily taking on a beta-sheet configuration and being linked by three hypervariable regions which form loops connecting and in some cases forming part of the beta-sheet structure. The hypervariable regions in each chain are held together by the FR immediately, and contribute to the formation of the antigen-binding site of the antibody with the hypervariable regions of the other chains (Kab., Sequences of Proteins of Immunological Interest, 5 th edition, Public Health service, National Institutes of Health, Bethesda, Md. (1991)), the constant domains do not directly determine the binding of the antibody to the antigen.

"antigen-binding fragment of an antibody" refers to a fragment, portion, region or domain of an antibody that is capable of binding to an epitope, and thus the terms "antigen-binding" and "epitope-binding" and "antigen-binding fragment of an antibody" are the same as "epitope-binding fragment of an antibody". Antigen-binding fragments may contain 1, 2, 3, 4, 5 or all 6 CDR domains of such antibodies and, although capable of binding to the epitope, may exhibit different specificities, affinities or selectivities.

Preferably, the antigen binding fragment contains all 6 CDR domains of the antibody.

An antigen-binding fragment of an antibody can be part of or comprise a single polypeptide chain (e.g., an scFv), or can be part of or comprise two or more polypeptide chains (each having an amino-terminus and a carboxy-terminus, e.g., a diabody, a Fab fragment, a Fab2 fragment, etc.).

IgG is an abbreviation of Immunoglobulin G (IgG), which is a major antibody component of serum, and human IgG has four subtypes based on the r-chain antigenic difference in IgG molecules: IgG1, IgG2, IgG3, IgG 4.

"monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts in individual antibodies. Monoclonal antibodies are highly specific, meaning directed against a single antigenic site. Furthermore, each monoclonal antibody is directed against a single determinant on the antigen, as opposed to a polyclonal antibody preparation comprising different antibodies directed against different determinants (epitopes). In addition to the specificity of a monoclonal antibody, a monoclonal antibody is advantageous in that it can be synthesized without contamination by other antibodies.

Host cells of the invention include, but are not limited to, E.coli, phage display systems, yeast, plant cells, animal cells.

The term "specifically binds" refers to an antibody or antigen-binding fragment thereof "specifically binding" a region of another molecule, specifically, reacting or binding to the epitope more frequently, more rapidly, with a longer duration, and/or with greater affinity or avidity relative to the other epitope. In some embodiments, an antibody or antigen-binding fragment thereof of the invention is present in an amount of at least 10^-7Affinity of M for binding antigen, e.g. 10^-8M、10^-9M、10^-10M、10^-11M or higher. Preferably, the antibody or antigen-binding fragment thereof binds under physiological conditions (e.g., in vivo). Thus, specifically binding to an antigen refers to the ability of the antibody or antigen-binding fragment thereof to bind to an antigen with the above-described specificity and/or under such conditions, and methods suitable for determining such binding are known in the art.

The term "combined" is generally meant to correspond to about 10^-6M or less, that is at least 10-fold, such as at least 100-fold, at least 1,000-fold less than the affinity of the antibody for binding to a non-specific antigen other than the designated antigen or a closely related antigen.

As used herein, the term "kd" (sec-1 or 1/s) refers to the off-rate constant for a particular antibody-antigen interaction, a value also known as k_offThe value is obtained.

As used herein, the term "ka" (M-1 x sec-1 or 1/Msec) refers to the knot of a particular antibody-antigen interactionSum rate constant, said value also being called k_onThe value is obtained. .

As used herein, the term "KD" (M) refers to the dissociation equilibrium constant for a particular antibody-antigen interaction and is obtained by dividing KD by ka.

As used herein, the term "KA" (M-1 or 1/M) refers to the binding equilibrium constant for a particular antibody-antigen interaction and is obtained by dividing KA by kd.

"treatment" includes, but is not limited to, one or more of the following assay characterizations: alleviating one or more symptoms caused by the disease; attenuation of the extent of disease; preventing or delaying the progression of the disease; preventing or delaying the spread of the disease; preventing or delaying the recurrence of the disease; delay or slow the progression of the disease; improving the disease condition; providing remission of the disease; reducing the dose of one or more other drugs required to treat the disease; delay of progression of the disease; increase or improve quality of life; increase body weight gain and/or prolong survival. In the present invention, "treatment" may be interpreted as a pathological consequence of cancer (e.g. reduction of tumor volume). Pharmaceutically acceptable carrier means a pharmaceutical carrier that is conventional in the pharmaceutical art, including but not limited to diluents, excipients, water, and the like; including but not limited to adhesives such as gelatin and polyvinylpyrrolidone; humectants such as glycerol; including but not limited to absorption enhancers such as quaternary ammonium compounds; including but not limited to surfactants such as cetyl alcohol, sodium lauryl sulfate, and the like.

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail below.