CN116574187B - Antibodies against GUCY2C and uses thereof - Google Patents
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- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
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Abstract
Antibodies against GUCY2C and uses thereof, particularly monoclonal antibodies against GUCY2C, bispecific antibodies against GUCY2C and CD3, nucleic acids comprising nucleotide sequences encoding the antibodies, vectors comprising the nucleic acids, and host cells comprising the nucleic acids or vectors, are disclosed herein. Pharmaceutical compositions and conjugates comprising the antibodies, and methods of treatment using the antibodies are also disclosed.
Description
Technical Field
The present invention relates to antibodies directed against GUCY2C, and the use of such antibodies, in particular their use in the treatment of cancer.
Background
Colorectal cancer (CRC) is one of the most frequently diagnosed cancers and is also a leading cause of cancer death worldwide. Guanylate cyclase 2C (GUCY 2C or GCC) belongs to the guanylate cyclase receptor family and has been reported to be expressed in a variety of cancers of the gastrointestinal tract, including more than 90% of CRC at each stage. GUCY2C is a brush border membrane receptor for the hormones guanosine and uridine, and for thermostable enterotoxins (STa) derived from E.coli. The expression of GUCY2C is limited to the luminal surface of healthy intestinal epithelium, and GUCY2C can provide promising target antigens for tumor-specific activation of T-cell redirection therapies administered systemically, due to tumor destruction of tight junctions. It was reported that the cell surface expression of GUCY2C was higher in medium to high differentiated tumors compared to low differentiated tumors.
High expression of GUCY2C in cancer cells such as CRC shows its potential ability for tumor therapy.
Disclosure of Invention
The present disclosure provides novel antibodies targeting GUCY2C or antigen binding fragments thereof, which may be in the form of monoclonal antibodies or bispecific antibodies, such as bispecific T cell cement (BiTE). The in vitro T cell dependent efficacy of antibodies has been evaluated for anti-tumor activity in vivo. The results of these functional assays demonstrate the potent anti-tumor effects of these engineered antibodies, particularly those in the form of BiTE.
In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to GUCY2C, comprising a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL comprises LCDR 1-3 having the amino acid sequences set forth in SEQ ID NOS: 1-3, respectively, and the VH comprises HCDR 1-3 having the amino acid sequences set forth in SEQ ID NOS: 6-8, respectively.
In some embodiments of the antibodies or antigen-binding fragments thereof disclosed herein, the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO. 4, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO. 9. In some embodiments, the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO. 11, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO. 12. In some embodiments, the VL comprises the amino acid sequence set forth in SEQ ID NO. 4 and the VH comprises the amino acid sequence set forth in SEQ ID NO. 9. In some embodiments, the VL comprises the amino acid sequence set forth in SEQ ID NO. 11 and the VH comprises the amino acid sequence set forth in SEQ ID NO. 12.
In some embodiments, the antibody is a murine antibodyA chimeric, humanized or human antibody. In some embodiments, the antibody belongs to an isotype selected from IgG, igA, igM, igE and IgD. In some embodiments, the antibody belongs to a subtype selected from the group consisting of IgG1, igG2, igG3, and IgG 4. In some embodiments, the antigen binding fragment is selected from the group consisting of Fab, fab ', F (ab') 2 Fv, scFv and ds-scFv.
In some embodiments, the antibody is a monoclonal antibody. In some embodiments, an antibody comprises a light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No. 5 and a heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No. 10.
In other embodiments, the antibody is a bispecific antibody or a multispecific antibody. In some embodiments, the antibody is a bispecific antibody further comprising a second antigen binding region that binds to a second antigen. In some embodiments, the second antigen is a tumor-associated antigen or an immune cell antigen. In some embodiments, the second antigen is a T cell antigen. In some embodiments, the T cell antigen is selected from the group consisting of T Cell Receptor (TCR), CD3, CD4, CD8, CD16, CD25, CD28, CD38, CD44, CD62L, CD69, ICOS, 41-BB (CD 137), and NKG2D.
In some embodiments, the second antigen is CD3 and the second antigen-binding region comprises a VL and a VH, wherein the VL comprises LCDR 1-3 having the amino acid sequences set forth in SEQ ID NOS 13-15, respectively, and the VH comprises HCDR 1-3 having the amino acid sequences set forth in SEQ ID NOS 17-19, respectively.
In some embodiments, the second antigen binding region comprises a VL and a VH, wherein the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No. 16, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID No. 20. In some embodiments, the second antigen binding region comprises a VL comprising the amino acid sequence set forth in SEQ ID NO. 16 and a VH comprising the amino acid sequence set forth in SEQ ID NO. 20.
In some embodiments, the antibody comprises an scFv comprising a VL and a VH of an antibody that specifically binds to GUCY2C, and the scFv is optionally linked to the N-terminus of the VL or VH of the second antigen binding region via a linker. In some embodiments, the antibody comprises: a first polypeptide chain comprising, from N-terminus to C-terminus: scFv, an optional linker, VL of the second antigen binding region, light chain constant region (CL), heavy chain constant region 2 (CH 2), and heavy chain constant region 3 (CH 3); and a second polypeptide chain comprising, from N-terminus to C-terminus: VH, heavy chain constant region 1 (CH 1), heavy chain constant region 2 (CH 2), and heavy chain constant region 3 (CH 3) of the second antigen binding region.
In some embodiments, the linker comprises an amino acid sequence selected from (G4S) n and GS (G4S) n, wherein n is an integer selected from 1-5, preferably the linker comprises an amino acid sequence as set forth in SEQ ID NO. 23 or 24.
In some embodiments, the first polypeptide chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO. 21, and the second polypeptide chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO. 22.
In some embodiments, the bispecific antibody is a bispecific T cell cement (BiTE).
In another aspect, the present disclosure provides a bispecific antibody or antigen-binding fragment thereof comprising a first antigen-binding region that binds to GUCY2C comprising a first light chain variable region (VL 1) and a first heavy chain variable region (VH 1), and a second antigen-binding region that binds to CD3 comprising a second light chain variable region (VL 2) and a second heavy chain variable region (VH 2), wherein VL1 comprises LCDR 1-3 each having an amino acid sequence as set forth in SEQ ID NOs 1-3; and VH1 comprises HCDR 1-3 having the amino acid sequences shown as SEQ ID NO. 6-8, respectively; and VL2 comprises LCDR 1-3 having the amino acid sequences shown in SEQ ID NOS 13-15, respectively; and VH2 comprises HCDR 1-3 having the amino acid sequences shown in SEQ ID NOS.17-19, respectively.
In some embodiments of the bispecific antibodies or antigen-binding fragments thereof disclosed herein, VL1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No. 4 and VH1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No. 9; and VL2 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO. 16 and VH2 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO. 20. In some embodiments, VL1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO. 11 and VH1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO. 12; and VL2 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO. 16 and VH2 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO. 20.
In some embodiments, VL1 comprises the amino acid sequence set forth in SEQ ID NO. 4 and VH1 comprises the amino acid sequence set forth in SEQ ID NO. 9; and VL2 comprises the amino acid sequence shown as SEQ ID NO. 16 and VH2 comprises the amino acid sequence shown as SEQ ID NO. 20. In some embodiments, VL1 comprises the amino acid sequence set forth in SEQ ID NO. 11 and VH1 comprises the amino acid sequence set forth in SEQ ID NO. 12; and VL2 comprises the amino acid sequence shown as SEQ ID NO. 16 and VH2 comprises the amino acid sequence shown as SEQ ID NO. 20.
In some embodiments, the first antigen binding region comprises an scFv comprising VL1 and VH1, and the scFv is optionally linked to the N-terminus of VL2 or VH2 via a linker. In some embodiments, the bispecific antibody comprises: a first polypeptide chain comprising, from N-terminus to C-terminus: scFv, optional linker, VL2, light chain constant region (CL), heavy chain constant region 2 (CH 2), and heavy chain constant region 3 (CH 3); and a second polypeptide chain comprising, from N-terminus to C-terminus: VH2, heavy chain constant region 1 (CH 1), heavy chain constant region 2 (CH 2), and heavy chain constant region 3 (CH 3).
In some embodiments, the linker comprises an amino acid sequence selected from (G4S) n and GS (G4S) n, wherein n is an integer selected from 1-5, preferably the linker comprises an amino acid sequence as set forth in SEQ ID NO. 23 or 24.
In some embodiments, the first polypeptide chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO. 21, and the second polypeptide chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO. 22.
In some embodiments, the bispecific antibody is a bispecific T cell cement (BiTE).
In yet another aspect, the present disclosure provides a nucleic acid comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof disclosed herein or a bispecific antibody or antigen-binding fragment thereof disclosed herein.
In another aspect, the present disclosure provides a vector comprising a nucleic acid disclosed herein.
In another aspect, the present disclosure provides a host cell comprising a nucleic acid disclosed herein or a vector disclosed herein.
In another aspect, the present disclosure provides a pharmaceutical composition comprising (i) an antibody or antigen-binding fragment thereof disclosed herein, or a bispecific antibody or antigen-binding fragment thereof disclosed herein, and (ii) a pharmaceutically acceptable carrier or excipient.
In some embodiments of the presently disclosed pharmaceutical compositions, the pharmaceutical composition further comprises a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from the group consisting of antibodies, chemotherapeutic agents, and small molecule drugs. In some embodiments, the second therapeutic agent is selected from the group consisting of a Bruton's Tyrosine Kinase (BTK) inhibitor, PI3K inhibitor, HDAC inhibitor, ERK inhibitor, MAPK inhibitor, PD-1 inhibitor, PD-L1 inhibitor, CTLA-4 inhibitor, TIGIT inhibitor, TIM3 inhibitor, VEGF inhibitor, LAG3 inhibitor, and glucocorticoid.
In yet another aspect, the present disclosure provides a conjugate comprising an antibody or antigen-binding fragment thereof disclosed herein or a bispecific antibody or antigen-binding fragment thereof disclosed herein, and a chemical moiety conjugated thereto.
In some embodiments of the conjugates disclosed herein, the chemical moiety may be selected from the group consisting of a therapeutic agent, a detectable moiety, and an immunostimulatory molecule.
In another aspect, the present disclosure provides a method of treating cancer in a subject comprising administering to the subject an effective amount of an antibody or antigen-binding fragment thereof disclosed herein, a bispecific antibody or antigen-binding fragment thereof disclosed herein, a pharmaceutical composition disclosed herein, or a conjugate disclosed herein.
In some embodiments of the methods disclosed herein, the cancer is a GUCY2C positive cancer. In some embodiments, the cancer is a digestive tract malignancy, preferably esophageal cancer or gastrointestinal cancer, such as colorectal cancer or gastric cancer.
In some embodiments, the method further comprises administering a second therapeutic agent to the subject. In some embodiments, the second therapeutic agent is selected from the group consisting of antibodies, chemotherapeutic agents, and small molecule drugs. In some embodiments, the second therapeutic agent is selected from the group consisting of a Bruton's Tyrosine Kinase (BTK) inhibitor, PI3K inhibitor, HDAC inhibitor, ERK inhibitor, MAPK inhibitor, PD-1 inhibitor, PD-L1 inhibitor, CTLA-4 inhibitor, TIGIT inhibitor, TIM3 inhibitor, VEGF inhibitor, LAG3 inhibitor, and glucocorticoid.
In another aspect, the present disclosure provides the use of an antibody or antigen-binding fragment thereof disclosed herein, a bispecific antibody or antigen-binding fragment thereof disclosed herein, a pharmaceutical composition disclosed herein, or a conjugate disclosed herein in the manufacture of a medicament for treating cancer in a subject.
In another aspect, the disclosure provides an antibody or antigen-binding fragment thereof disclosed herein, a bispecific antibody or antigen-binding fragment thereof disclosed herein, a pharmaceutical composition disclosed herein, or a conjugate disclosed herein for use in treating cancer in a subject.
In some embodiments of the uses disclosed herein, the cancer is a GUCY2C positive cancer. In some embodiments, the cancer is a digestive tract malignancy, preferably esophageal cancer or gastrointestinal cancer, such as colorectal cancer or gastric cancer. In some embodiments, an antibody or antigen-binding fragment thereof disclosed herein, a bispecific antibody or antigen-binding fragment thereof disclosed herein, a pharmaceutical composition disclosed herein, or a conjugate disclosed herein is associated with a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from the group consisting of antibodies, chemotherapeutic agents, and small molecule drugs. In some embodiments, the second therapeutic agent is selected from the group consisting of a Bruton's Tyrosine Kinase (BTK) inhibitor, PI3K inhibitor, HDAC inhibitor, ERK inhibitor, MAPK inhibitor, PD-1 inhibitor, PD-L1 inhibitor, CTLA-4 inhibitor, TIGIT inhibitor, TIM3 inhibitor, VEGF inhibitor, LAG3 inhibitor, and glucocorticoid.
Drawings
An appreciation of the features and advantages of the present invention can be obtained by reference to the following detailed description that sets forth exemplary embodiments, which utilize the principles of the present invention, and the accompanying drawings thereof:
figure 1 shows the binding of anti-GUCY 2C mAb to recombinant human GUCY2C as measured by ELISA.
FIG. 2 shows ADCC killing of anti-GUCY 2C mAbs against GUCY2C positive HT1080-GUCY2C cells in the presence of Jurkat-CD16a cells.
FIG. 3A shows the binding of GUCY2C×CD3 IBiTE to recombinant human GUCY2C as measured by ELISA.
Figure 3B shows the binding of GUCY2c×cd3ibite to recombinant human CD3 as measured by ELISA.
FIG. 3C shows the co-binding of GUCY2C×CD3 IBiTE to recombinant human GUCY2C and CD3 as measured by ELISA.
Fig. 4A shows the binding of GUCY2c×cd3 IBiTE to GUCY2C positive tumor cell line HT55 as measured by flow cytometry.
Fig. 4B shows the binding of GUCY2c×cd3 IBiTE to GUCY2C positive tumor cell line SW948 as measured by flow cytometry.
FIG. 4C shows the binding of GUCY2C×CD3 IBiTE to the GUCY2C positive tumor cell line LS174T-GUCY2C as measured by flow cytometry.
FIG. 5A shows GUCY2C X CD3 IBiTE induced T cell activation in the presence of the GUCY2C positive cell line HT 55.
FIG. 5B shows GUCY2C X CD3 IBiTE induced T cell activation in the presence of the GUCY2C positive cell line SW 948.
FIG. 5C shows GUCY2C X CD3 IBiTE induced T cell activation in the presence of the GUCY2C positive cell line LS174T-GUCY 2C.
Figure 6A shows the killing of HT55 cells by GUCY2c×cd3 IBiTE in the presence of human PBMC.
Figure 6B shows killing of SW948 cells by GUCY2c×cd3 IBiTE in the presence of human PBMC.
FIG. 6C shows the killing of GUCY2C X CD3 IBiTE against LS174T-GUCY2C cells in the presence of human PBMC.
FIG. 7A shows tumor volumes in B-NDG mice xenografted with HT1080-GUCY2C/PBMC treated with GUCY2C X CD3 IBiTE. Mice treated with PBS served as negative controls. Data represent mean tumor volume±sem.
FIG. 7B shows body weight of B-NDG mice xenografted with HT1080-GUCY2C/PBMC treated with GUCY2C X CD3 IBiTE. Mice treated with PBS served as negative controls. Data represent mean body weight ± SEM.
Detailed Description
The above features and advantages of the present invention and additional features and advantages thereof will be more clearly understood from the following detailed description of embodiments taken in conjunction with the accompanying drawings.
The embodiments described herein with reference to the drawings are illustrative, exemplary, and are intended for general understanding of the present invention. The embodiments should not be construed as limiting the scope of the invention. The same or similar elements and elements having the same or similar functions are denoted by the same reference numerals throughout the specification.
Unless otherwise indicated or defined, all terms used have the ordinary meaning in the art as would be apparent to one of ordinary skill. For example, refer to standard manuals such as Leuenberger, H.G.W, nagel, B.and Klbl, H.eds., "A multilingual glossary of biotechnological terms (IUPAC Recommendations)", helvetica Chimica Acta (1995), CH-4010 Basel, switzerland; sambrook et al, "Molecular Cloning: A Laboratory Manual" (2 nd Ed.), vols.1-3, cold Spring Harbor Laboratory Press (1989); F.Ausubel et al, eds., "Current protocols in molecular biology", green Publishing and Wiley InterScience, new York (1987); roitt et al, "Immunology (6 th Ed.)," Mosby/Elsevier, edinburgh (2001); and Janeway et al, "Immunology" (6 th Ed.; garland Science Publishing/Churchill Livingstone, new York (2005)), and the general background art cited above.
Definition of the definition
As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an antibody" includes a plurality of antibodies and, in some embodiments, reference to "an antibody" includes a plurality of antibodies, and so forth.
Unless otherwise indicated or defined, the terms "comprises," "comprising," and variations thereof such as "comprises" and "comprising" are to be understood to imply the inclusion of a stated element or step or group of elements or steps but not the exclusion of any other element or step or group of elements or steps.
As used herein, the term "antibody" refers to an immunoglobulin molecule that has the ability to specifically bind to a particular antigen. Such molecules typically comprise two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (or domain) (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2 and CH 3. Each light chain consists of a light chain variable region (or domain) (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain CL. The variable regions of the heavy and light chains of antibodies contain binding domains that interact with the antigen. The constant region of an antibody may mediate the binding of an immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and components of the complement system such as C1q (the first component of the classical pathway of complement activation).
The heavy chain of an immunoglobulin can be divided into three functional regions: fd region, hinge region and Fc region (crystallizable fragment). The Fd region comprises VH and CH1 domains and binds to the light chain to form Fab (antigen binding fragment). The Fc fragment is responsible for immunoglobulin effector functions including, for example, complement fixation and binding to cognate Fc receptors of effector cells. The hinge region found in the IgG, igA and IgD immunoglobulin classes acts as a flexible spacer region, allowing the Fab portion to move freely in space relative to the Fc region. Hinge domains are structurally diverse, with sequence and length varying between immunoglobulin classes and subclasses.
According to crystallographic studies, the immunoglobulin hinge region can be further subdivided in structure and function into three regions: upper, core and lower hinges (Shin et al Immunological Reviews 130:87, 1992). The upper hinge includes the amino acid from the carboxy terminus of CH1 to the first residue in the hinge that limits movement, typically the first cysteine residue that forms an interchain disulfide bond between the two heavy chains. The length of the upper hinge region is related to the fragment flexibility of the antibody. The core hinge region contains inter-heavy chain disulfide bonds. The lower hinge region is attached to the amino terminus of the CH2 domain and includes residues in the CH2 domain. Conformational changes allowed by the structure and flexibility of the immunoglobulin hinge region polypeptide sequence may affect the effector function of the Fc portion of the antibody.
The "light chain variable region" (VL) or "heavy chain variable region" (VH) consists of "framework" regions separated by three "complementarity determining regions" or "CDRs". The framework regions are used to align CDRs that specifically bind to an epitope. CDRs include amino acid residues in antibodies that are primarily responsible for antigen binding. The VL domain and VH domain both comprise the following Framework (FR) and CDR regions from amino-to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. CDR1, CDR2, and CDR3 of the VL domain are also referred to herein as LCDR1, LCDR2, and LCDR3, respectively; CDR1, CDR2, and CDR3 of the VH domain are also referred to herein as HCDR1, HCDR2, and HCDR3, respectively.
The amino acid arrangement of each VL domain and VH domain is consistent with any conventional definition of CDRs. Conventional definitions include the Kabat definition (Kabat, sequences of Proteins of Immunological Interest (National Institutes of Health, bethesda, MD, 1987 and 1991)), the Chothia definition (Chothia and Lesk, J. Mol. Biol. 196:901-917, 1987; chothia et al Nature 342:878-883, 1989); chothia Kabat CDR, wherein CDR-H1 is a complex of Chothia CDR and Kabat CDR; abM definition used by Oxford Molecular antibody modeling software; CONTACT definition by Martin et al (world wide web bioinfo. Org. Uk/abs). Kabat provides a widely used numbering convention (Kabat numbering system) in which corresponding residues between different heavy chains or between different light chains are given the same number. The present disclosure may use CDRs defined according to any of these numbering systems, but preferred embodiments use Kabat-defined CDRs.
Immunoglobulin molecules can be divided into five classes (isotypes) based on the amino acid sequence of the antibody heavy chain constant region: igA, igD, igE, igG and IgM, and can be further divided into different subtypes such as IgG1, igG2, igG3, igG4, igA1, igA2, etc. Based on the amino acid sequence of the light chain, the light chain of an antibody can be divided into lambda (λ) chains and kappa (κ) chains.
As used herein, the term "antibody" is to be understood in its broadest sense and includes monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, antibody fragments, and multispecific antibodies (e.g., bispecific antibodies) that contain at least two antigen-binding regions. Antibodies may contain additional modifications such as non-naturally occurring amino acids, mutations in the Fc region, and mutations in glycosylation sites. Antibodies also include post-translationally modified antibodies, fusion proteins containing an epitope of an antibody, and any other modified immunoglobulin molecule containing an antigen recognition site, so long as the antibodies exhibit the desired biological activity.
As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies. That is, each antibody that makes up the population is identical except for a small number of mutations that may occur naturally. Monoclonal antibodies are highly specific and directed against a single antigen. The term "monoclonal antibody" herein is not limited to antibodies produced by hybridoma technology, nor should it be construed as requiring antibodies produced by any particular method.
The term "bispecific antibody" is in the context of the present invention to be understood as an antibody having two different antigen binding regions defined by different antibody sequences. This is understood to be binding to different targets, but also includes binding to different epitopes of one target. The term "bispecific antibody" as used herein is to be understood in its broadest sense and includes full length bispecific antibodies and antigen binding fragments thereof. Bispecific antibodies may contain additional modifications such as non-naturally occurring amino acids, mutations in the Fc region, and mutations in glycosylation sites. Bispecific antibodies also include post-translationally modified antibodies, fusion proteins containing an epitope of an antibody, and any other modified immunoglobulin molecule containing an antigen recognition site, so long as the antibodies exhibit the desired biological activity.
As used herein, the term "antigen-binding fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen. It has been shown that the antigen binding function of an antibody can be performed by fragments of full length antibodies.
Examples of antigen binding fragments encompassed in the term "antigen binding portion" of an antibody encompass: (i) A Fab fragment, a monovalent fragment consisting of VL, VH, CL and CH1 domains; (ii) F (ab') 2 A fragment comprising a bivalent fragment of two Fab fragments linked by a disulfide bond at the hinge region; (iii) Fab' fragments, which are essentially Fab with a partial hinge region; (iv) Fd fragment consisting of VH and CH1 domains; (v) Fd' fragments having VH and CH1 domainsOne or more cysteine residues at the C-terminus of the CH1 domain; (vi) Fv fragment consisting of VL and VH domains of the antibody single arm; (vii) a dAb fragment consisting of a VH domain; (viii) an isolated Complementarity Determining Region (CDR); (ix) Nanobodies, heavy chain variable regions containing a single variable domain and two constant domains. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined, using recombinant methods, by a synthetic linker, enabling them to be made into a single protein chain, in which the VL and VH regions pair to form monovalent molecules, known as single chain Fv (scFv). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" of an antibody. In addition, the term also includes "linear antibodies" comprising a pair of tandem Fd fragments (VH-CH 1-VH-CH 1), which together with a complementary light chain polypeptide form an antigen binding region, as well as modified versions of any of the foregoing fragments that retain antigen binding activity.
These antigen binding fragments can be obtained using conventional techniques known to those skilled in the art and the fragments screened for utility in the same manner as whole antibodies.
As used herein, the term "binding" or "specific binding" refers to a non-random binding reaction between two molecules, such as an antibody and its target antigen. The binding specificity of an antibody may be determined based on affinity and/or avidity. Affinity represents the equilibrium constant (KD) for antigen to antibody dissociation and is a measure of the strength of binding between an epitope and the antigen binding site of an antibody: the smaller the value of KD, the stronger the binding strength between the epitope and the antibody. Alternatively, affinity can also be expressed as an affinity constant (KA), which is 1/KD.
Avidity is a measure of the strength of binding between an antibody and the associated antigen. Avidity relates to the affinity between an epitope and the antigen binding site of an antibody and the number of relevant binding sites present on the antibody. Typically, an antibody will bind an antigen with the following dissociation constants (KD): 10 -5 M to 10 -12 M or less, and preferably 10 -7 M to 10 -12 M or less, and more preferably 10 -8 M to 10 -12 M, and/or haveThere are the following binding affinities: at least 10 7 M -1 Preferably at least 10 8 M -1 More preferably at least 10 9 M -1 Such as at least 10 12 M -1 . Generally considered to be any greater than 10 -4 K of M D Values represent non-specific binding. Specific binding of an antibody to an antigen or antigenic determinant can be determined in any known suitable manner, including, for example, scatchard analysis and/or competitive binding assays, such as Radioimmunoassays (RIA), enzyme Immunoassays (EIA) and sandwich competition assays, as well as different variants thereof known in the art.
The term "epitope" refers to the site on an antigen to which an antibody binds. Epitopes can be formed by contiguous amino acids or by non-contiguous amino acids juxtaposed by tertiary folding of one or more proteins. Epitopes formed by consecutive amino acids (also referred to as linear epitopes) are typically retained in exposure to denaturing solvents, whereas epitopes formed by tertiary folding (also referred to as conformational epitopes) are typically lost in treatment with denaturing solvents. Epitopes typically comprise at least 3, more typically at least 5 or 8-10 amino acids in a unique spatial conformation. The epitope defines the smallest binding site of an antibody and is therefore a specific target for the antibody or antigen binding fragment thereof.
As used herein, the term "sequence identity" refers to the degree to which two sequences (amino acids) have identical residues at identical positions after alignment. For example, "an amino acid sequence is Y X% identical" refers to an amino acid sequence that is X% identical to SEQ ID NO: Y and is described as having X% of the residues in the amino acid sequence identical to the residues of the sequence disclosed in SEQ ID NO: Y. Typically, such calculations are performed using a computer program. Exemplary procedures for comparing and aligning pairs of sequences include ALIGN (Myers and Miller, 1988), FASTA (Pearson and Lipman, 1988; pearson, 1990), gapped BLAST (Altschul et al, 1997), BLASTP, BLASTN or GCG (Devereux et al, 1984).
Furthermore, in determining the degree of sequence identity between two amino acid sequences, the skilled artisan may consider so-called "conservative" amino acid substitutions, which may generally be described as amino acid substitutions in which an amino acid residue is replaced with another amino acid residue having a similar chemical structure, which have little or no effect on the function, activity, or other biological properties of the polypeptide. Such conservative amino acid substitutions are well known in the art.
Such conservative substitutions are preferably substitutions in which one amino acid in the following groups (a) to (e) is substituted by another amino acid residue in the same group: (a) small aliphatic, non-polar or weakly polar residues: ala, ser, thr, pro and Gly; (b) Polar, negatively charged residues and (uncharged) amides: asp, asn, glu and Gln; (c) polar, positively charged residues: his, arg and Lys; (d) large aliphatic, nonpolar residues: met, leu, ile, val and Cys; and (e) an aromatic residue: phe, tyr and Trp.
Particularly preferred conservative substitutions are as follows: ala to Gly or to Ser; arg to Lys; asn to Gln or to His; asp to Glu; cys to Ser; gln to Asn; glu to Asp; gly to Ala or to Pro; his to Asn or to Gln; lie to Leu or to Val; leu to Ile or to Val; lys to Arg, to gin, or to Glu; met to Leu, to Tyr or to Ile; phe to Met, to Leu, or to Tyr; ser to Thr; thr to Ser; trp to Tyr; tyr to Trp; and/or Phe to Val, to Ile or to Leu.
As used herein, the term "tumor-associated antigen" refers to an antigen that is differentially expressed in cancer cells as compared to normal cells, and thus can be used to target cancer cells.
As used herein, the term "CD3" refers to a human CD3 protein complex having 5 peptide chains, a gamma chain, a delta chain, an epsilon chain, a zeta chain, and a eta chain, and associating with T cell receptors alpha and beta chains to form a TCR-CD3 complex. The term includes any CD3 variant, subtype and species homolog that may be expressed naturally by cells including T cells or by cells transfected with a gene or cDNA encoding the above chain.
As used herein, the term "bispecific T cell cement" or "BiTE" refers to a polypeptide chain molecule having two antigen binding domains, one of which binds to a T cell antigen and the second of which binds to an antigen presented on the surface of a target cell (see PCT publication WO 05/061547; baeuerle et al, 2008,Drugs of the Future 33:137-147; barbou et al, 2008,Science 321:974-977, which is incorporated herein by reference in its entirety). Thus, the BiTE of the present disclosure has an antigen-binding region that binds to the GUCY2C and a second antigen-binding region that targets a T cell antigen.
As used herein, the term "vector" is intended to mean a nucleic acid molecule capable of transporting another nucleic acid to which it is linked.
As used herein, the term "host cell" refers to a cell into which an expression vector has been introduced.
The term "pharmaceutically acceptable" means that the carrier or excipient is compatible with the other ingredients of the composition and not substantially deleterious to the recipient thereof, and/or that such carrier or excipient is approved or otherwise available for inclusion in a pharmaceutical composition for parenteral administration to a human.
As used herein, the terms "treatment," "therapy," "treatment," and the like refer to administration of an agent or procedure for the purpose of achieving an effect. These effects may be prophylactic in terms of completely or partially preventing a disease or symptom thereof, and/or may be therapeutic in terms of achieving a partial or complete cure of the disease and/or disease symptom. As used herein, "treating" may include treating a disease or disorder (e.g., cancer) in a mammal, particularly a human, and includes: (a) preventing the occurrence of a disease or disease symptom in a subject who may be susceptible to the disease (e.g., including a disease that may be associated with or caused by a primary disease) but has not been diagnosed with the disease, (b) inhibiting the disease, i.e., arresting its development, and (c) alleviating the disease, i.e., causing regression of the disease. Treatment may refer to any indication of success in the treatment or amelioration or prevention of cancer, including any objective or subjective parameter, such as reduction of symptoms; relief; elimination of disease symptoms or making disease conditions more tolerable to the patient; slowing the rate of deterioration or decay; or to attenuate the final node of the deterioration. Treatment or amelioration of symptoms is based on one or more objective or subjective parameters; including the results of the physician's examination. Thus, the term "treatment" includes administration of an antibody or composition or conjugate disclosed herein to prevent or delay, alleviate, or prevent or inhibit the development of symptoms or disorders associated with a disease (e.g., cancer). The term "therapeutic effect" refers to the reduction, elimination or prevention of a disease, disease symptom or disease side effect in a subject.
As used herein, the term "effective amount" refers to an amount administered to a subject to treat a disease sufficient to effect treatment of the disease.
As used herein, the term "subject" refers to any mammalian subject for whom diagnosis, treatment or therapy is desired. "mammal" for therapeutic purposes refers to any animal classified as a mammal, including humans, domestic animals, and laboratory, zoo, sports, or pet animals, such as dogs, horses, cats, cattle, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys, etc.
anti-GUCY 2C antibodies
The present disclosure provides antibodies or antigen-binding fragments thereof that specifically bind to GUCY2C, comprising a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL comprises LCDR 1-3 having the amino acid sequences set forth in SEQ ID NOS: 1-3, respectively, and the VH comprises HCDR 1-3 having the amino acid sequences set forth in SEQ ID NOS: 6-8, respectively.
In some embodiments, CDR sequences are defined according to the Kabat numbering system.
When CDR sequences are defined according to the Kabat numbering system, the VL of the antibodies disclosed herein comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences shown as SEQ ID NO: 1 (SASSSVSYIH), SEQ ID NO: 2 (STSNLAS) and SEQ ID NO: 3 (QQRSSYPLT), respectively, and the VH of the antibodies disclosed herein comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequences shown as SEQ ID NO: 6 (SYAMT), SEQ ID NO: 7 (TMSSGGGYTYYLDSVKG) and SEQ ID NO: 8 (HNYGYNYAMDY), respectively.
In some embodiments, the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO. 4, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO. 9. In other embodiments, the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO. 11, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO. 12.
In some embodiments, the VL comprises a functional variant of the amino acid sequence shown as SEQ ID NO. 4 or 11, formed by insertion, deletion and/or substitution of one or more amino acids therein, provided that the functional variant retains the ability to bind GUCY 2C. In some embodiments, the VH comprises a functional variant of the amino acid sequence shown as SEQ ID NO 9 or 12, formed by insertion, deletion and/or substitution of one or more amino acids therein, provided that the functional variant retains the ability to bind GUCY 2C.
The functional variant comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to the amino acid sequence of the parent polypeptide.
In the context of functional variants, the number of amino acids inserted, deleted and/or substituted preferably does not exceed 40%, more preferably does not exceed 35%, more preferably is 1% to 33%, and more preferably is 5% to 30%, more preferably is 10% to 25%, more preferably is 15% to 20% of the total number of amino acids in the parent amino acid sequence. For example, the number of inserted, deleted and/or substituted amino acids may be 1 to 20, preferably 1 to 10, more preferably 1 to 7, still more preferably 1 to 5, most preferably 1 to 2. In preferred embodiments, the number of amino acids inserted, deleted and/or substituted is 1, 2, 3, 4, 5, 6 or 7.
In some embodiments, insertions, deletions, and/or substitutions may be made in the Framework (FR) region, e.g., in FR1, FR2, FR3, and/or FR 4.
In some embodiments, the substitution of one or more amino acids may be conservative substitutions of one or more amino acids. Such conservative substitutions are preferably substitutions in which one amino acid in the following groups (a) to (e) is substituted by another amino acid residue in the same group: (a) small aliphatic, non-polar or weakly polar residues: ala, ser, thr, pro and Gly; (b) Polar, negatively charged residues and (uncharged) amides: asp, asn, glu and Gln; (c) polar, positively charged residues: his, arg and Lys; (d) large aliphatic, nonpolar residues: met, leu, he, val and Cys; and (e) an aromatic residue: phe, tyr and Trp.
Particularly preferred conservative substitutions are as follows: ala to Gly or to Ser; arg to Lys; asn to Gln or to His; asp to Glu; cys to Ser; gln to Asn; glu to Asp; gly to Ala or to Pro; his to Asn or to Gln; lie to Leu or to Val; leu to Ile or to Val; lys to Arg, to gin, or to Glu; met to Leu, to Tyr or to Ile; phe to Met, to Leu, or to Tyr; ser to Thr; thr to Ser; trp to Tyr; tyr to Trp; and/or Phe to Val, to Ile or to Leu.
In a preferred embodiment, the VL comprises the amino acid sequence shown as SEQ ID NO. 4 and the VH comprises the amino acid sequence shown as SEQ ID NO. 9. In another preferred embodiment, the VL comprises the amino acid sequence shown as SEQ ID NO. 11 and the VH comprises the amino acid sequence shown as SEQ ID NO. 12.
In some embodiments, the antibody is a murine antibody, chimeric antibody, humanized antibody, or human antibody.
Immunoglobulin molecules can be divided into five classes (isotypes) based on the amino acid sequence of the antibody heavy chain constant region: igA, igD, igE, igG and IgM, and can be further divided into different subtypes such as IgG1, igG2, igG3, igG4, igA1, igA2, etc. Based on the amino acid sequence of the light chain, the light chain of an antibody can be divided into lambda (λ) chains and kappa (κ) chains. The antibodies disclosed herein may be of any of the classes or subtypes described above.
In some embodiments, the antibody belongs to an isotype selected from IgG, igA, igM, igE and IgD. In some embodiments, the antibody belongs to a subtype selected from the group consisting of IgG1, igG2, igG3, and IgG 4. In a preferred embodiment, the antibody is an IgG1 antibody.
The antibodies disclosed herein may be whole antibodies or antigen-binding fragments thereof. The antigen binding fragment may be any fragment of an antibody that retains the ability to specifically bind to GUCY 2C. Examples of antigen binding fragments include, but are not limited to: fab fragments; f (ab') 2 fragments; fab' fragments; fd fragment; fd' fragment; fv fragments; an scFv fragment; a dAb fragment; isolated Complementarity Determining Regions (CDRs); a nanobody; linear antibodies consisting of a pair of Fd fragments in tandem (VH-CH 1-VH-CH 1), and modified versions of any of the foregoing fragments that retain antigen-binding activity.
In some embodiments, the antigen binding fragment is selected from the group consisting of Fab, fab ', F (ab') 2 Fv, scFv and ds-scFv. In a preferred embodiment, the antigen binding fragment is a Fab. In another preferred embodiment, the antigen binding fragment is an Fv. In another preferred embodiment, the antigen binding fragment is an scFv.
In some embodiments, the antibody is a monoclonal antibody. In some embodiments, an antibody comprises a light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No. 5 and a heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No. 10.
In some embodiments, the light chain comprises a functional variant of the amino acid sequence shown as SEQ ID NO. 5, formed by insertion, deletion and/or substitution of one or more amino acids therein, provided that the functional variant retains the ability to bind to GUCY 2C. In some embodiments, the heavy chain comprises a functional variant of the amino acid sequence shown as SEQ ID NO. 10, formed by insertion, deletion and/or substitution of one or more amino acids therein, provided that the functional variant retains the ability to bind to GUCY 2C.
The functional variant comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to the amino acid sequence of the parent polypeptide.
In some embodiments, the number of amino acids inserted, deleted and/or substituted preferably does not exceed 40%, more preferably does not exceed 35%, more preferably is 1% to 33%, and more preferably is 5% to 30%, more preferably is 10% to 25%, more preferably is 15% to 20% of the total number of amino acids in the parent amino acid sequence. For example, the number of inserted, deleted and/or substituted amino acids may be 1 to 50, preferably 1 to 20, more preferably 1 to 10, still more preferably 1 to 5. In preferred embodiments, the number of amino acids inserted, deleted and/or substituted is 1, 2, 3, 4, 5, 6 or 7.
In some embodiments, insertions, deletions, and/or substitutions may be in the Framework (FR) regions, such as FR1, FR2, FR3, and/or FR4; and/or constant regions, such as CL, CH1, CH2, and/or CH 3.
In some embodiments, the substitution of one or more amino acids may be conservative substitutions of one or more amino acids. Examples of conservative substitutions are described above.
In a preferred embodiment, the antibody comprises a light chain comprising the amino acid sequence shown as SEQ ID NO. 5 and a heavy chain comprising the amino acid sequence shown as SEQ ID NO. 10.
In other embodiments, the antibody is a bispecific antibody or a multispecific antibody. In some embodiments, the antibody is a bispecific antibody further comprising a second antigen binding region that binds a second antigen. In some embodiments, the second antigen is a tumor-associated antigen or an immune cell antigen.
A number of tumor-associated antigens have been identified in the art as being associated with a particular cancer. In some embodiments, the tumor-associated antigen is an antigen that can elicit a distinct tumor-specific immune response. Some of these antigens are encoded by, but not necessarily expressed by, normal cells. These antigens can be characterized as antigens that are normally silenced (i.e., not expressed) in normal cells, antigens that are expressed only at certain stages of differentiation, and antigens that are expressed over time, such as embryonic and fetal antigens. Other cancer cell antigens are encoded by mutant cell genes such as oncogenes (e.g., activated ras oncogenes), suppressor genes (e.g., P53 mutants), and fusion proteins resulting from internal deletions or chromosomal translocations. Other cancer antigens may be encoded by viral genes such as those carried by RNA and DNA oncolytic viruses. Many other tumor-associated antigens and antibodies thereto are known and/or commercially available and may also be prepared by those skilled in the art.
Examples of tumor-associated antigens include, but are not limited to, 5T4, alpha fetoprotein, CA-125, carcinoembryonic antigen, CD19, CD20, CD22, CD23, CD30, CD33, CD40, CD56, CD79, CD78, CD123, CD138, c-Met, CSPG4, igM, AXL, EGFR, EGFRvIII, epithelial tumor antigen, ERBB2, FLT3, folate binding protein, GD2, GD3, HIV-1 envelope glycoprotein gp41, HIV-1 envelope glycoprotein gp120, melanoma-associated antigen, MUC-1, mutated p53, mutated ras, ROR1, GPC3, VEGFR2, and combinations thereof.
In some embodiments, the second antigen is a T cell antigen. In some embodiments, the T cell antigen is selected from the group consisting of T Cell Receptor (TCR), CD3, CD4, CD8, CD16, CD25, CD28, CD38, CD44, CD62L, CD69, ICOS, 41-BB (CD 137), and NKG2D, or any combination thereof. In some embodiments, the T cell antigen is CD3 and the second antigen binding region binds to any one of the gamma, delta, epsilon, zeta and eta chains of CD 3.
In some embodiments, the second antigen is CD3 and the second antigen-binding region comprises a VL and a VH, wherein the VL comprises LCDR 1-3 having the amino acid sequences set forth in SEQ ID NOS 13-15, respectively, and the VH comprises HCDR 1-3 having the amino acid sequences set forth in SEQ ID NOS 17-19, respectively.
In some embodiments, CDR sequences are defined according to the Kabat numbering system. When using a CDR sequence as defined by Kabat, the VL of the second antigen binding region disclosed herein comprises LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in SEQ ID NO: 13 (RSSTGAVTTSNYAN), SEQ ID NO: 14 (GANKRAP) and SEQ ID NO: 15 (ALWYSNLWV), respectively, and the VH of the second antigen binding region disclosed herein comprises HCDR1, HCDR2 and HCDR3 having amino acid sequences as shown in SEQ ID NO: 17 (TYANN), SEQ ID NO: 18 (RIRSKYNNYATYYADSVKG) and SEQ ID NO: 19 (HGNFGSSYVSYFAY), respectively.
In some embodiments, the second antigen binding region comprises a VL and a VH, wherein the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No. 16, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID No. 20.
In some embodiments, the VL comprises a functional variant of the amino acid sequence shown as SEQ ID NO. 16, formed by insertion, deletion and/or substitution of one or more amino acids therein, provided that the functional variant retains the ability to bind CD 3. In some embodiments, the VH comprises a functional variant of the amino acid sequence shown as SEQ ID NO. 20, formed by insertion, deletion and/or substitution of one or more amino acids therein, provided that the functional variant retains the ability to bind CD 3.
In some embodiments, the number of amino acids inserted, deleted and/or substituted preferably does not exceed 40%, more preferably does not exceed 35%, more preferably is 1% to 33%, and more preferably is 5% to 30%, more preferably is 10% to 25%, more preferably is 15% to 20% of the total number of amino acids in the parent amino acid sequence. For example, the number of inserted, deleted and/or substituted amino acids may be 1 to 20, preferably 1 to 10, more preferably 1 to 7, still more preferably 1 to 5, most preferably 1 to 2. In preferred embodiments, the number of amino acids inserted, deleted and/or substituted is 1, 2, 3, 4, 5, 6 or 7.
In some embodiments, insertions, deletions, and/or substitutions may be made in the Framework (FR) region, e.g., in FR1, FR2, FR3, and/or FR 4.
In some embodiments, the substitution of one or more amino acids may be conservative substitutions of one or more amino acids. Examples of conservative substitutions are described above.
In a preferred embodiment, the second antigen binding region comprises a VL comprising the amino acid sequence set forth in SEQ ID NO. 16 and a VH comprising the amino acid sequence set forth in SEQ ID NO. 20.
In some embodiments, the antibody comprises an scFv comprising a VL and a VH of an antibody that specifically binds to GUCY2C, and the scFv is optionally linked to the N-terminus of the VL or VH of the second antigen binding region via a linker. In some embodiments, the antibody comprises: a first polypeptide chain comprising, from N-terminus to C-terminus: scFv, an optional linker, VL of the second antigen binding region, light chain constant region (CL), heavy chain constant region 2 (CH 2), and heavy chain constant region 3 (CH 3); and a second polypeptide chain comprising, from N-terminus to C-terminus: VH, heavy chain constant region 1 (CH 1), heavy chain constant region 2 (CH 2), and heavy chain constant region 3 (CH 3) of the second antigen binding region.
In some embodiments, the linker comprises an amino acid sequence selected from (G4S) n and GS (G4S) n, wherein n is an integer selected from 1-5, preferably the linker comprises an amino acid sequence as set forth in SEQ ID NO. 23 or 24.
In some embodiments, the first polypeptide chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO. 21, and the second polypeptide chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO. 22. In a preferred embodiment, the first polypeptide chain comprises the amino acid sequence shown as SEQ ID NO. 21 and the second polypeptide chain comprises the amino acid sequence shown as SEQ ID NO. 22.
In some embodiments, the bispecific antibody is a bispecific T cell cement (BiTE). In some embodiments, the bispecific antibody is in the form of HBiTE, as described in PCT application No. PCT/US2018/016524 (which is incorporated herein by reference in its entirety). In HBiTE, the light chain comprises, from N-terminus to C-terminus, an anti-target VL domain, an anti-CD 3 VL-CL, and a monomeric human IgG1 Fc (e.g., mfc 7.2); and the heavy chain comprises, from the N-terminus to the C-terminus, an anti-target VH domain, an anti-CD 3 VH-CH1, and a monomeric human IgG1 Fc (e.g., mfc 7.2). Monomer fc7.2 contains two amino acid mutations (T366L and Y407H) that inhibit Fc homodimerization.
Bispecific antibodies
The present disclosure provides bispecific antibodies or antigen-binding fragments thereof comprising a first antigen-binding region that binds to GUCY2C comprising a first light chain variable region (VL 1) and a first heavy chain variable region (VH 1), and a second antigen-binding region that binds to CD3 comprising a second light chain variable region (VL 2) and a second heavy chain variable region (VH 2), wherein VL1 comprises LCDR 1-3 each having an amino acid sequence as set forth in SEQ ID NOs 1-3; and VH1 comprises HCDR 1-3 having the amino acid sequences shown as SEQ ID NO. 6-8, respectively; and VL2 comprises LCDR 1-3 having the amino acid sequences shown in SEQ ID NOS 13-15, respectively; and VH2 comprises HCDR 1-3 having the amino acid sequences shown in SEQ ID NOS.17-19, respectively.
In some embodiments, CDR sequences are defined according to the Kabat numbering system.
When CDR sequences are defined according to the Kabat numbering system, VL1 of the bispecific antibody disclosed herein comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences as shown in SEQ ID No. 1 (SASSSVSYIH), SEQ ID No. 2 (stsnas) and SEQ ID No. 3 (QQRSSYPLT), respectively, and VH1 of the bispecific antibody disclosed herein comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequences as shown in SEQ ID No. 6 (SYAMT), SEQ ID No. 7 (TMSSGGGYTYYLDSVKG) and SEQ ID No. 8 (HNYGYNYAMDY), respectively, VL2 of the bispecific antibody disclosed herein comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences as shown in SEQ ID No. 13 (RSSTGAVTTSNYAN), SEQ ID No. 14 (gank) and SEQ ID No. 15 (ALWYSNLWV), respectively, and VH2 of the bispecific antibody disclosed herein comprises HCDR1, HCDR3 having the amino acid sequences as shown in SEQ ID No. 17 (tymn), SEQ ID No. 18 (RIRSKYNNYATYYADSVKG) and SEQ ID No. 19 (HCDR 3).
In some embodiments of the bispecific antibodies or antigen-binding fragments thereof disclosed herein, VL1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No. 4 and VH1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No. 9; and VL2 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO. 16 and VH2 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO. 20. In some embodiments, VL1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO. 11 and VH1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO. 12; and VL2 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO. 16 and VH2 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO. 20.
In some embodiments, VL1 comprises a functional variant of an amino acid sequence as set forth in SEQ ID NO. 4 or 11, formed by insertion, deletion and/or substitution of one or more amino acids therein, provided that the functional variant retains the ability to bind GUCY 2C. In some embodiments, VH1 comprises a functional variant of the amino acid sequence shown as SEQ ID NO 9 or 12, formed by insertion, deletion and/or substitution of one or more amino acids therein, provided that the functional variant retains the ability to bind GUCY 2C. In some embodiments, VL2 comprises a functional variant of the amino acid sequence shown as SEQ ID NO. 16, formed by insertion, deletion and/or substitution of one or more amino acids therein, provided that the functional variant retains the ability to bind CD 3. In some embodiments, VH2 comprises a functional variant of the amino acid sequence shown as SEQ ID NO. 20, formed by insertion, deletion and/or substitution of one or more amino acids therein, provided that the functional variant retains the ability to bind CD 3.
The functional variant comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to the amino acid sequence of the parent polypeptide.
In the context of functional variants, the number of amino acids inserted, deleted and/or substituted preferably does not exceed 40%, more preferably does not exceed 35%, more preferably is from 1% to 33%, more preferably is from 5% to 30%, more preferably is from 10% to 25%, more preferably is from 15% to 20% of the total number of amino acids in the parent amino acid sequence. For example, the number of inserted, deleted and/or substituted amino acids may be 1 to 20, preferably 1 to 10, more preferably 1 to 7, still more preferably 1 to 5, most preferably 1 to 2. In preferred embodiments, the number of amino acids inserted, deleted and/or substituted is 1, 2, 3, 4, 5, 6 or 7.
In some embodiments, insertions, deletions, and/or substitutions may be made in the Framework (FR) region, e.g., in FR1, FR2, FR3, and/or FR 4.
In some embodiments, the substitution of one or more amino acids may be conservative substitutions of one or more amino acids. Examples of conservative substitutions are described above.
In a preferred embodiment, VL1 comprises the amino acid sequence shown as SEQ ID NO. 4 and VH1 comprises the amino acid sequence shown as SEQ ID NO. 9; and VL2 comprises the amino acid sequence shown as SEQ ID NO. 16 and VH2 comprises the amino acid sequence shown as SEQ ID NO. 20. In another preferred embodiment, VL1 comprises the amino acid sequence shown as SEQ ID NO. 11 and VH1 comprises the amino acid sequence shown as SEQ ID NO. 12; and VL2 comprises the amino acid sequence shown as SEQ ID NO. 16 and VH2 comprises the amino acid sequence shown as SEQ ID NO. 20.
In some embodiments, the first antigen binding region comprises an scFv comprising VL1 and VH1, and the scFv is optionally linked to the N-terminus of VL2 or VH2 via a linker. In some embodiments, the scFv is optionally linked to the N-terminus of VL2 via a linker. In some embodiments, the scFv is optionally linked to the N-terminus of VH2 via a linker. In some embodiments, the scFv is formed by linking VL1 and VH1 via a linker.
In some embodiments, the joint may be any flexible joint. In some embodiments, the linker comprises an amino acid sequence of (G4S) n, wherein n is an integer selected from 1-5. In some embodiments, the linker may comprise the amino acid sequence GGGGS (SEQ ID NO: 25). In some embodiments, the linker may comprise the amino acid sequence GGGGSGGGGS (SEQ ID NO: 26). In some embodiments, the linker may comprise the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 23). In some embodiments, the linker may comprise the amino acid sequence GGGGSGGGGSGGGGSGGGGGGS (SEQ ID NO: 27). In some embodiments, the linker may comprise the amino acid sequence GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 28). In a preferred embodiment, the linker comprises the amino acid sequence shown as SEQ ID NO. 23.
In other embodiments, the linker comprises the amino acid sequence of GS (G4S) n, wherein n is an integer selected from 1-5. In some embodiments, the linker may comprise the amino acid sequence GSGGGGS (SEQ ID NO: 29). In some embodiments, the linker may comprise the amino acid sequence GSGGGGSGGGGS (SEQ ID NO: 24). In some embodiments, the linker may comprise the amino acid sequence GSGGGGSGGGGSGGGGS (SEQ ID NO: 30). In some embodiments, the linker may comprise the amino acid sequence GSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 31). In some embodiments, the linker may comprise the amino acid sequence GSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 32). In a preferred embodiment, the linker comprises the amino acid sequence shown as SEQ ID NO. 24.
Bispecific antibodies disclosed herein may comprise an Fc region comprising CH2 and CH3 of the antibody.
The Fc region may be of any isotype including, but not limited to, igG1, igG2, igG3, and IgG4, and may contain one or more mutations or modifications. In one embodiment, the Fc region is or is derived from an IgG1 isotype, optionally with one or more mutations or modifications. In another embodiment, the Fc region is or is derived from an IgG4 isotype, optionally with one or more mutations or modifications. In one embodiment, the Fc region is a human IgG1 Fc.
In one embodiment, the Fc region has reduced effector function, e.g., reduced ADCC, ADCP, CDC and/or Clq, fcγri, fcγrii, or fcγriiia binding. For example, the Fc region may be of the IgGl isotype, or of a non-IgGl isotype, e.g., igG2, igG3, or IgG4, that has been mutated such that the ability to mediate effector functions is reduced or even eliminated. Such mutations have been described, for example, in Dall' Acqua WF et al, J Immunol 177 (2): 1129-1138 (2006) and Hezareh M, J Virol.; 75 (24): 12161-12168 (2001). For example, the Fc region may comprise an amino acid sequence having one or more of the following amino acid substitutions as compared to the wild-type sequence: E233P, L234A, L F, L235A, L235E, G237A, N297A, N297D, P331S and P329G.
In one embodiment, the Fc region comprises a mutation that removes an Asn-linked glycosylated receptor site or a mutation that is otherwise manipulated to alter the glycosylation characteristics. For example, in the IgG1 Fc region, the Asn-linked glycosylation site can be removed using the N297Q mutation. Thus, in a specific embodiment, the Fc region comprises an IgG1 sequence having the N297Q mutation.
In a further embodiment, the Fc region is glycoengineered to reduce fucose and thus enhance ADCC, for example by adding a compound to the medium during antibody production, as described in US2009317869 or as described in van Berkel et al (2010) biotechnol. Bioeng. 105:350, or by knocking out cells using FUT8, for example as described in Yamane-Ohnuki et al (2004) biotechnol. Bioeng 87:614. Alternatively, ADCC may be optimized using the method described by Uma ñ a et al (1999) Nature Biotech 17:176. In another embodiment, the Fc region is engineered to enhance complement activation, for example as described in Natsume et al (2009) Cancer sci.100:2411.
In some embodiments, the Fc region comprises modifications or mutations that can inhibit Fc homodimerization. In some embodiments, the Fc region comprises a variant of a human IgG1 Fc wild-type sequence. The variant may comprise amino acid substitutions at positions (Kabat numbering) of human IgG 1T 366 and Y407. Preferably, T366 is substituted with L (leucine). Preferably, Y407 is substituted with I (isoleucine), F (phenylalanine), L (leucine), M (methionine), H (histidine), K (lysine), S (serine), Q (glutamine), T (threonine), W (tryptophan), a (alanine), G (glycine), or N (asparagine). More preferably, Y407 is substituted with H. In one embodiment, T366 is substituted with L and Y407 is substituted with H.
In some embodiments, the Fc region may be a monomeric human IgG1 Fc (e.g., mfc 7.2), as described in PCT application No. PCT/US2018/016524, which is incorporated herein by reference in its entirety.
In some embodiments, the bispecific antibody comprises: a first polypeptide chain comprising, from N-terminus to C-terminus: scFv, optional linker, VL2, light chain constant region (CL), heavy chain constant region 2 (CH 2), and heavy chain constant region 3 (CH 3); and a second polypeptide chain comprising, from N-terminus to C-terminus: VH2, heavy chain constant region 1 (CH 1), heavy chain constant region 2 (CH 2), and heavy chain constant region 3 (CH 3).
The bispecific antibodies disclosed herein may also comprise a hinge region of the antibody.
The hinge region of an IgG class antibody refers to the short amino acid sequence region between the CH1 and CH2 portions of the heavy chain, which is relatively flexible in the natural state of the antibody. The hinge region may comprise part or all of the wild-type hinge sequence or a variant thereof having one or more substitutions.
In some embodiments, the bispecific antibody comprises: a first polypeptide chain comprising, from N-terminus to C-terminus: scFv, optional linker, VL2, light chain constant region (CL), hinge region, heavy chain constant region 2 (CH 2), and heavy chain constant region 3 (CH 3); and a second polypeptide chain comprising, from N-terminus to C-terminus: VH2, heavy chain constant region 1 (CH 1), hinge region, heavy chain constant region 2 (CH 2), and heavy chain constant region 3 (CH 3).
In some embodiments, the CH1, CH2, CH3 and hinge regions are each independently derived from an immunoglobulin isotype IgG (e.g., human IgG), preferably from IgG subtypes selected from IgG1, igG2 and IgG4 (e.g., human IgG1, igG2 and IgG 4). In some embodiments, CL is derived from a lambda light chain or a kappa light chain.
In some embodiments, one or both of CH2 comprises at least one amino acid mutation capable of reducing the effector function of a bispecific antibody. For example, CH2 may comprise at least one amino acid substitution selected from E233P, L234A, L234F, L235A, L235E, G237A, N297A, N297D, P S and P329G or any combination thereof. In some embodiments, the at least one mutation is selected from L234A, L235A, G237A, P329G or any combination thereof. In a preferred embodiment, the at least one mutation is selected from L234A, L235A, G a and P329G. In some embodiments, the at least one mutation is selected from L234F, L235E, P329G or a combination thereof. In a preferred embodiment, the at least one mutation is selected from L234F, L235E and P329G.
In some embodiments, one or both of CH3 comprises at least one amino acid mutation capable of reducing homodimerization between the first and second polypeptide chains. In a preferred embodiment, amino acid T366 of one or both of CH3 is substituted with L (leucine), and amino acid Y407 of one or both of CH3 is substituted with I (isoleucine), F (phenylalanine), L (leucine), M (methionine), H (histidine), K (lysine), S (serine), Q (glutamine), T (threonine), W (tryptophan), a (alanine), G (glycine), or N (asparagine). In one embodiment, amino acid T366 of one or both of CH3 is substituted with L and amino acid Y407 of one or both of CH3 is substituted with H. In a preferred embodiment, in CH3 of both the first and second polypeptide chains, T366 is substituted with L and Y407 is substituted with H.
The linkers may be those as described above. For example, the joint may be any flexible joint. In some embodiments, the linker comprises an amino acid sequence selected from (G4S) n and GS (G4S) n, wherein n is an integer selected from 1-5, preferably the linker comprises an amino acid sequence as set forth in SEQ ID NO. 23 or 24.
In some embodiments, the first polypeptide chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO. 21, and the second polypeptide chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO. 22.
In some embodiments, the first polypeptide chain comprises a functional variant of the amino acid sequence shown as SEQ ID NO. 21, formed by insertion, deletion and/or substitution of one or more amino acids therein, provided that the functional variant retains the ability to bind GUCY2C and CD 3. In some embodiments, the second polypeptide chain comprises a functional variant of the amino acid sequence shown as SEQ ID NO. 22, formed by insertion, deletion and/or substitution of one or more amino acids therein, the precursor being a functional variant that retains the ability to bind CD 3.
The functional variant comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to the amino acid sequence of the parent polypeptide.
In some embodiments, the number of amino acids inserted, deleted and/or substituted preferably does not exceed 40%, more preferably does not exceed 35%, more preferably is 1% to 33%, and more preferably is 5% to 30%, more preferably is 10% to 25%, more preferably is 15% to 20% of the total number of amino acids in the parent amino acid sequence. For example, the number of inserted, deleted and/or substituted amino acids may be 1 to 50, preferably 1 to 20, more preferably 1 to 10, still more preferably 1 to 5. In preferred embodiments, the number of amino acids inserted, deleted and/or substituted is 1, 2, 3, 4, 5, 6 or 7.
In some embodiments, insertions, deletions, and/or substitutions may be in the Framework (FR) regions, such as FR1, FR2, FR3, and/or FR4; and/or constant regions, such as CL, CH1, CH2, and/or CH 3.
In some embodiments, the substitution of one or more amino acids may be conservative substitutions of one or more amino acids. Examples of conservative substitutions are described above.
In a preferred embodiment, the first polypeptide chain comprises the amino acid sequence shown as SEQ ID NO. 21; and the second polypeptide chain comprises the amino acid sequence shown as SEQ ID NO. 22.
In some embodiments, the bispecific antibody is a bispecific T cell cement (BiTE).
Nucleic acid
The present disclosure provides nucleic acids comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof disclosed herein or a bispecific antibody or antigen-binding fragment thereof disclosed herein.
The term "nucleic acid" includes single-and double-stranded nucleotide polymers. The nucleic acid may be a ribonucleotide or a deoxyribonucleotide or a modified form of either type of nucleotide. Such modifications include base modifications such as bromouridine and inosine derivatives, ribose modifications such as 2',3' -dideoxyribose, internucleotide linkage modifications such as phosphorothioates, phosphorodithioates, phosphoroselenos, phosphorodiselenos, phosphorophenylthioates, phosphoroanilide, and phosphoramidates.
For example, the invention provides nucleic acid molecules encoding any of the heavy chain variable region sequences disclosed herein. The invention also provides nucleic acid molecules that are at least 90%, at least 95%, at least 98%, or at least 99% identical to a nucleic acid encoding any of the heavy chain variable region sequences disclosed herein.
For example, the invention provides nucleic acid molecules encoding any of the light chain variable region sequences disclosed herein. The invention also provides nucleic acid molecules that are at least 90%, at least 95%, at least 98%, or at least 99% identical to a nucleic acid encoding any of the light chain variable region sequences disclosed herein.
For example, the invention provides nucleic acid molecules encoding heavy chain variable region sequences comprising CDR sequences of any one of the heavy chain variable region sequences disclosed herein. The invention also provides nucleic acid molecules encoding heavy chain variable region sequences comprising CDR sequences that are at least 90%, at least 95%, at least 98%, or at least 99% identical to CDR sequences of any one of the heavy chain variable region sequences disclosed herein.
For example, the invention provides nucleic acid molecules encoding light chain variable region sequences comprising CDR sequences of any one of the light chain variable region sequences disclosed herein. The invention also provides nucleic acid molecules encoding light chain variable region sequences comprising CDR sequences that are at least 90%, at least 95%, at least 98%, or at least 99% identical to CDR sequences of any one of the light chain variable region sequences disclosed herein.
In some embodiments, the nucleic acid is ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). In some embodiments, the invention provides ribonucleic acid (RNA) comprising a nucleotide sequence encoding an antibody disclosed herein. In some embodiments, the invention provides deoxyribonucleic acid (DNA) comprising a deoxynucleotide sequence encoding an antibody disclosed herein.
In some embodiments, deoxyribonucleic acid (DNA) may be introduced into human cells in vivo. In some embodiments, the deoxyribonucleic acid (DNA) of the invention is contained in a carrier or delivery agent. In some embodiments, the deoxyribonucleic acid (DNA) of the invention is integrated into the genome of a cell.
In some embodiments, ribonucleic acid (RNA) can be introduced into human cells in vivo. In some embodiments, ribonucleic acid (RNA) of the invention is contained in a vector or delivery agent.
Carrier body
The present disclosure provides vectors comprising the nucleic acids disclosed herein.
In some embodiments, the vector is an expression vector capable of expressing a polypeptide comprising the heavy or light chain variable region of an antibody. For example, the invention provides expression vectors comprising any of the nucleic acid molecules described above.
Any carrier may be suitable for use in the present disclosure. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a retroviral vector, a DNA vector, a murine leukemia virus vector, an SFG vector, a plasmid, an RNA vector, an adenovirus vector, a baculovirus vector, an Epstein Barr virus vector, a papovavirus vector, a vaccinia virus vector, a herpes simplex virus vector, an adenovirus-associated vector (AAV), a lentiviral vector, or any combination thereof. Suitable exemplary vectors include, for example, pGAR, pBABE-Puro, pBABE-neo largeTcDNA, pBABE-hygro-hTERT, pMKO.1 GFP, MSCV-IRES-GFP, pMSCV PIG (puroIRES GFP empty plasmid), pMSCV-loxp-dsRed-loxp-eGFP-Puro-WPRE, MSCV IRES luciferase, pMIG, MDH1-PGK-GFP_2.0, ttRMPVIR, pMSCV-IRES-mCherry FP, pRetrox GFP T2A Cre, pRXTN, pLncEXP, and pLXIN-Luc.
The expression vector may be any suitable recombinant expression vector. Suitable vectors include vectors designed for proliferation and amplification or for expression or both, such as plasmids and viruses. For example, the vector may be selected from the pUC series (Fermentas Life Sciences, glen Burnie, md.), the pBluescript series (Stratagene, laJolla, calif.), the pET series (Novagen, madison, wis.), the pGEX series (Pharmacia Biotech, uppsala, sweden) and the pEX series (Clontech, palo Alto, calif.). Phage vectors such as λGT10, λGT11, λ ZapII (Stratagene), λEMBL4, and λNM1149 can also be used. Examples of plant expression vectors useful in the present disclosure include pBI01, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). Examples of animal expression vectors useful in the present disclosure include pcDNA, pEUK-Cl, pMAM and pMAMneo (Clontech).
Recombinant expression vectors can be prepared using standard recombinant DNA techniques described, for example, in Sambrook et al, molecular Cloning: A Laboratory Manual, 3rd ed., cold Spring Harbor Press, cold Spring Harbor, N.Y. 2001, and Ausubel et al, current Protocols in Molecular Biology, greene Publishing Associates and John Wiley & Sons, NY, 1994. Circular or linear expression vector constructs can be prepared to contain the replication system functions in prokaryotic or eukaryotic host cells. Replication systems may be derived from, for example, COLEL, 2 μ plasmid, λ, SV40, bovine papilloma virus, etc.
For example, the vector may be an adenovirus vector comprising a nucleotide sequence encoding an antibody disclosed herein. The vector may be administered to a subject in vivo and then into cells of the subject, thereby integrating the nucleotide sequences encoding antibodies disclosed herein into the genome of the cells, which then express the antibodies disclosed herein.
Host cells
The present disclosure provides host cells comprising a nucleic acid disclosed herein or a vector disclosed herein.
Any cell can be used as a host cell for the nucleic acids or vectors of the present disclosure. In some embodiments, the cell may be a prokaryotic cell, a fungal cell, a yeast cell, or a higher eukaryotic cell such as a mammalian cell. Suitable prokaryotic cells include, but are not limited to, eubacteria, such as gram-negative bacteria Or gram-positive organisms, e.g. of the enterobacteriaceae familyEnterobactehaceae) Such as Escherichia genusEscherichia) For example Escherichia coli [ ]E. coli) The method comprises the steps of carrying out a first treatment on the surface of the Enterobacter genusEnterobacter) The method comprises the steps of carrying out a first treatment on the surface of the Erwinia genusErwinia) The method comprises the steps of carrying out a first treatment on the surface of the Klebsiella genusKlebsiella) The method comprises the steps of carrying out a first treatment on the surface of the Proteus genus ]Proteus) The method comprises the steps of carrying out a first treatment on the surface of the Salmonella genusSalmonella) For example Salmonella typhimurium @Salmonella typhimurium) The method comprises the steps of carrying out a first treatment on the surface of the Serratia genusSerratia) For example Serratia marcescens @Serratia marcescans) Shigella species @ HeShigella) The method comprises the steps of carrying out a first treatment on the surface of the Bacillus genusBacilli) Such as bacillus subtilis @B. subtilis) And Bacillus licheniformisB. licheniformis) The method comprises the steps of carrying out a first treatment on the surface of the Pseudomonas genusPseudomonas) Such as Pseudomonas aeruginosaP. aeruginosa) The method comprises the steps of carrying out a first treatment on the surface of the And Streptomyces genusStreptomyces). In some embodiments, the cell is a human cell. In some embodiments, the cell is an immune cell. In some embodiments, host cells include, for example, CHO cells, such as CHOs cells and CHO-K1 cells, or HEK293 cells, such as HEK293A, HEK293T and HEK293FS.
The host cells of the invention are prepared by introducing the vectors disclosed herein or the nucleic acids disclosed herein in vitro or ex vivo. The host cells of the invention can be administered to a subject and express the antibodies disclosed herein in vivo.
The present invention provides host cells into which any of the above vectors have been introduced. The invention also provides a method of producing an antibody of the invention, the method comprising a) culturing a host cell of the fourth aspect of the invention under conditions suitable for production of the antibody; and b) obtaining the antibody from the culture.
Pharmaceutical composition
The present disclosure provides pharmaceutical compositions comprising an antibody or antigen-binding fragment thereof disclosed herein, or a bispecific antibody or antigen-binding fragment thereof disclosed herein, and a pharmaceutically acceptable carrier or excipient.
The antibodies or antigen-binding fragments or agents thereof (also referred to herein as "active compounds") of the invention, and derivatives, fragments, analogs, and homologs thereof, may be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise an antibody or antigen-binding fragment thereof or a pharmaceutical agent, and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Preferred examples of such carriers or excipients include, but are not limited to, water, saline, ringer's solution, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils can also be used. The use of such media and medicaments for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, it is contemplated that it will be used in the composition. Supplementary active compounds may also be incorporated into the compositions.
In some embodiments, the pharmaceutical composition further comprises a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from the group consisting of antibodies, chemotherapeutic agents, and small molecule drugs. In some embodiments, the second therapeutic agent is selected from the group consisting of a Bruton's Tyrosine Kinase (BTK) inhibitor, PI3K inhibitor, HDAC inhibitor, ERK inhibitor, MAPK inhibitor, PD-1 inhibitor, PD-L1 inhibitor, CTLA-4 inhibitor, TIGIT inhibitor, TIM3 inhibitor, VEGF inhibitor, LAG3 inhibitor, and glucocorticoid.
In some embodiments, the therapeutic agent is a chemotherapeutic agent. Chemotherapeutic agents may include, for example, cytotoxic agents, antimetabolites (e.g., folic acid antagonists, purine analogs, pyrimidine analogs, etc.), topoisomerase inhibitors (e.g., camptothecin derivatives, anthraquinones, anthracyclines, epipodophyllotoxins, quinoline alkaloids, etc.), antimicrotubule agents (e.g., taxanes, vinca alkaloids), protein synthesis inhibitors (e.g., cephalotaxine, camptothecin derivatives, quinoline alkaloids), alkylating agents (e.g., alkyl sulfonates, aziridines, nitrogen mustards, nitrosoureas, platinum derivatives, triazenes, etc.), alkaloids, terpenoids, and kinase inhibitors.
The pharmaceutical compositions of the present invention may be formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral administration, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions for parenteral, intradermal, or subcutaneous application may include the following components: sterile diluents, such as water for injection, saline solutions, fixed oils, polyethylene glycols, glycerol, propylene glycol or other synthetic solvents; antimicrobial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as tetraethylammonium oxalate (EDTA); buffers such as acetate, citrate or phosphate; and agents for modulating tonicity, such as sodium chloride or dextrose. The pH can be adjusted with an acid or base, such as hydrochloric acid or sodium hydroxide. Parenteral formulations may be enclosed in ampules, disposable syringes or multiple dose vials made of glass or plastic.
Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (if water-soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, cremophor EL TM (BASF, parippany, n.j.) or Phosphate Buffered Saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy injection is possible. It must be stable under the conditions of preparation and storage and must be protected from the contaminating action of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycols, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. The action of microorganisms can be prevented by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is preferred to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. By including agents in the composition that delay absorption, such as aluminum monostearate and gelatin, the injectable set can be prolongedAbsorption of the compound.
The sterile injectable solution may be prepared by: the desired amount of active compound is admixed (as required) with one or a combination of the ingredients listed above in an appropriate solvent and then sterilized by filtration. Typically, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation are vacuum drying and freeze-drying which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
Oral compositions typically include an inert diluent or an edible carrier. They may be enclosed in gelatin capsules or compressed into tablets. For the purposes of oral therapeutic administration, the active compounds may be mixed with excipients and used in the form of tablets, troches or capsules. Oral compositions may also be prepared using a fluid carrier that serves as a mouthwash, wherein the compounds in the fluid carrier are administered orally and rinsed and expectorated or swallowed. Pharmaceutically compatible binding agents and/or adjuvant materials may be included as part of the composition. Tablets, pills, capsules, troches and the like may contain any of the following ingredients or compounds of similar nature: binding agents, such as microcrystalline cellulose, tragacanth or gelatin; excipients, such as starch or lactose, disintegrants, such as alginic acid, primogel or corn starch; lubricants, such as magnesium stearate or Sterotes; glidants, such as colloidal silicon dioxide; sweeteners, such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
For inhalation administration, the compounds are delivered in the form of an aerosol spray from a pressure vessel or dispenser or nebulizer containing a suitable propellant (e.g., a gas such as carbon dioxide).
Systemic administration may also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, detergents for transmucosal administration, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated as ointments, salves, gels or creams as known in the art.
The active compounds can also be formulated in the form of suppositories (e.g., using conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
In one embodiment, the active compound is prepared with a carrier (e.g., a controlled release formulation, including implants and microencapsulated delivery systems) that will protect the compound from rapid elimination from the body. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid may be used. Methods of preparing such formulations will be apparent to those skilled in the art.
The present invention provides therapeutic compositions comprising an antibody or antigen-binding fragment thereof of the invention. The therapeutic compositions according to the present invention will be administered with suitable carriers, excipients, and other agents incorporated into the formulation to provide improved transfer, delivery, tolerability, etc. Many suitable formulations can be found in all prescriptions known to pharmaceutical chemists: remington's Pharmaceutical Sciences, mack Publishing Company, easton, PA. Such formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, vesicle-containing lipids (cationic or anionic) (e.g., LIPOFECTIN ™), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, polyethylene glycol emulsions (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing polyethylene glycols. See also Powell et al, "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52:52:238-311.
Conjugate(s)
The present disclosure provides conjugates comprising an antibody or antigen-binding fragment thereof disclosed herein or a bispecific antibody or antigen-binding fragment thereof disclosed herein, and a chemical moiety conjugated thereto.
In the context of the present disclosure, a "conjugate" is an antibody or antibody fragment (e.g., an antigen binding fragment) covalently linked to a chemical moiety. The chemical moiety may be, for example, a drug, toxin, therapeutic agent, detectable label, protein, nucleic acid, lipid, nanoparticle, carbohydrate, or recombinant virus. Antibody conjugates are commonly referred to as "immunoconjugates". When the conjugate comprises an antibody linked to a drug (e.g., a cytotoxic agent), the conjugate is commonly referred to as an "antibody-drug conjugate" or "ADC".
The term "conjugation" or "linking" may refer to making two polypeptides into one continuous polypeptide molecule. In one embodiment, the antibody is linked to a chemical moiety. In another embodiment, the antibody linked to the chemical moiety is further linked to a lipid or other molecule to a protein or peptide to increase its half-life in vivo. The ligation may be performed chemically or recombinantly. In one embodiment, the linkage is chemical, wherein the reaction between the antibody moiety and the chemical moiety produces a covalent bond formed between the two molecules to form one molecule. Peptide linkers (short peptide sequences) may optionally be included between the antibody and the chemical moiety.
The chemical moiety may be attached to the antibodies of the invention using any number of means known to those skilled in the art. Covalent and non-covalent attachment means may be used. The procedure for attaching the chemical moiety to the antibody varies depending on the chemical structure of the chemical moiety. Polypeptides typically contain a variety of functional groups; such as carboxylic acid (COOH), free amine (-NH 2), or sulfhydryl (-SH) moieties, which may be used to react with suitable functional groups on an antibody to result in the binding of chemical moieties. Alternatively, the antibody is derivatized to expose or attach additional reactive functional groups. Derivatization may involve attachment of any of a number of known linker molecules. The linker may be any molecule for linking the antibody to the chemical moiety. The linker is capable of forming a covalent bond with both the antibody and the chemical moiety. Suitable linkers are well known to those skilled in the art and include, but are not limited to, straight or branched chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. Where the antibodies and chemical moieties are polypeptides, the linker may be attached to the constituent amino acids (e.g., via disulfide bonds to cysteines) or to the alpha carbon amino and carboxyl groups of the terminal amino acids through their pendant groups.
In some cases, it is desirable to release the chemical moiety from the antibody when the immunoconjugate reaches its target site. Thus, in these cases, the immunoconjugate will comprise a cleavable linkage near the target site.
Conditions to which the enzymatically active or immunoconjugate is subjected within or near the target cell may promote cleavage of the linker to release the chemical moiety from the antibody.
In view of the numerous methods reported for attaching various radiodiagnostic compounds, radiotherapeutic compounds, markers (such as enzymes or fluorescent molecules), drugs, toxins and other agents to antibodies, one skilled in the art will be able to determine the appropriate method of attaching a given agent to an antibody or other polypeptide.
The antibodies disclosed herein can be derivatized or linked to another molecule (e.g., another peptide or protein). Typically, the antibody or portion thereof is derivatized such that binding to the target antigen is not adversely affected by derivatization or labeling. For example, an antibody may be functionally linked (by chemical coupling, genetic fusion, non-covalent association, or other means) to one or more other molecular entities, such as another antibody (e.g., a bispecific or bivalent antibody), a detection agent, an agent, and/or a protein or peptide that may mediate the association of an antibody or antibody portion with another molecule (e.g., a streptavidin core region or a polyhistidine tag).
One type of derivatized antibody is produced by cross-linking two or more antibodies (of the same type or different types). Suitable crosslinking agents include heterobifunctional or homobifunctional crosslinking agents (e.g., disuccinimidyl suberate) having two distinct reactive moieties (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) separated by a suitable spacer. Such linkers are commercially available.
In some embodiments of the presently disclosed conjugates, the chemical moiety is selected from the group consisting of a therapeutic agent, a detectable moiety, and an immunostimulatory molecule.
In some embodiments, the therapeutic agent includes, but is not limited to, an immunomodulatory agent, a radioactive compound, an enzyme (e.g., perforin), a chemotherapeutic agent (e.g., cisplatin), or a toxin. In some embodiments, the therapeutic agent may be, for example, maytansine, geldanamycin, a tubulin inhibitor such as a tubulin binding agent (e.g., an auristatin) or a minor groove binding agent such as calicheamicin.
Other suitable therapeutic agents include, for example, small molecule cytotoxic agents, i.e., compounds having a molecular weight less than 700 daltons that have the ability to kill mammalian cells. Such compounds may also contain toxic metals capable of cytotoxic effects. In addition, it is understood that these small molecule cytotoxic agents also include prodrugs, i.e., compounds that decompose or transform under physiological conditions to release the cytotoxic agent. Examples of such agents include cisplatin, maytansine derivatives, lazithromycin, calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodium photosensitizing element II, temozolomide, topotecan, trimethoprim, orestatin E vincristine, and doxorubicin; peptide cytotoxins, i.e., proteins or fragments thereof that have the ability to kill mammalian cells, such as ricin, diphtheria toxin, pseudomonas bacterial exotoxin A, DNA enzyme, and rnase; radionuclides, i.e., unstable isotopes of elements that decay with the simultaneous emission of one or more a or β particles or gamma rays, such as iodine-131, rhenium-186, indium-111, yttrium-90, bismuth-210, bismuth-213, actinium-225, and astatine-213; chelating agents can be used to facilitate the binding of these radionuclides to molecules or their multimers.
In some embodiments, the detectable moiety may be selected from biotin, streptavidin, an enzyme or a catalytically active fragment thereof, a radionuclide, a nanoparticle, a paramagnetic metal ion, or a fluorescent, phosphorescent, or chemiluminescent molecule. Detectable moieties for diagnostic purposes include, for example, fluorescent labels, radiolabels, enzymes, nucleic acid probes, and contrast agents.
The antibody may be conjugated to a detectable label; for example, detectable labels that can be detected by ELISA, spectrophotometry, flow cytometry, microscopy, or diagnostic imaging techniques such as Computed Tomography (CT), computed Axial Tomography (CAT) scan, magnetic Resonance Imaging (MRI), magnetic resonance imaging NMRI), magnetic resonance tomography (MTR), ultrasound, fiber optic examination, and laparoscopy. Specific, non-limiting examples of detectable labels include fluorophores, chemiluminescent agents, enzymatic linkages, radioisotopes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for detection by MRI). For example, useful detectable labels include fluorescent compounds including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors, and the like. Bioluminescent labels, such as luciferase, green Fluorescent Protein (GFP) and Yellow Fluorescent Protein (YFP), may also be used.
The antibody or antigen binding fragment may also be conjugated to enzymes useful for detection, such as horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase, glucose oxidase, and the like. When the antibody or antigen binding fragment is conjugated to a detectable enzyme, it can be detected by adding additional reagents that the enzyme uses to produce a recognizable reaction product. For example, when horseradish peroxidase reagent is present, the addition of hydrogen peroxide and diaminobenzidine can result in a colored reaction product, which can be detected visually. Antibodies or antigen binding fragments can also be conjugated to biotin and detected by indirect measurement of avidin or streptavidin binding. It should be noted that avidin itself may be conjugated to an enzyme or fluorescent label.
The antibody may be fused to a self-labeling protein tag (e.g., haloTag). For example, a protein tag may be cloned into the end of the constant region. HaloTag is a self-labeling protein tag derived from bacterial enzymes (haloalkane dehalogenases) intended to be covalently bound to synthetic ligands. In some cases, the synthetic ligands comprise a chloroalkane linker attached to a fluorophore, such as a near infrared fluorophore (Los et al (2008) ACS Chem biol 3 (6): 373-82).
The antibodies may be labeled with a magnetic agent such as gadolinium. Antibodies may also be labeled with lanthanides (e.g., europium and dysprosium) and manganese.
Paramagnetic particles such as superparamagnetic iron oxide may also be used as labels. The antibody may also be labeled with a predetermined polypeptide epitope (e.g., leucine zipper pair sequence, binding site of a second antibody, metal binding domain, epitope tag) recognized by a second reporter gene. In some embodiments, the tags are attached by spacer arms of various lengths to reduce potential steric hindrance.
Antibodies may also be labeled with radiolabeled amino acids. Radiolabels may be used for diagnostic and therapeutic purposes. For example, radiolabels may be used to detect expression of target antigens by X-ray, emission spectroscopy, or other diagnostic techniques. Examples of polypeptide labels include, but are not limited to, the following radioisotopes or radionucleotides: 3H, 14C, 15N, 35S, 90Y, 99Tc, 111In, 125I, 131I.
In some embodiments, the immunostimulatory molecule is an immune effector molecule that stimulates an immune response. For example, the immunostimulatory molecules may be cytokines such as IL-2 and IFN-gamma, chemokines such as IL-8, platelet factor 4, melanoma growth stimulatory proteins, complement activators; viral/bacterial protein domains, or viral/bacterial peptides.
Therapeutic method
The present disclosure provides methods of treating cancer in a subject comprising administering to the subject an effective amount of an antibody or antigen-binding fragment thereof disclosed herein, a bispecific antibody or antigen-binding fragment thereof disclosed herein, a pharmaceutical composition disclosed herein, or a conjugate disclosed herein.
In some embodiments of the methods disclosed herein, the cancer is a GUCY2C positive cancer. In some embodiments, the cancer is a digestive tract malignancy. In some embodiments, the cancer is a gastrointestinal cancer. Examples of cancers include, but are not limited to, esophageal cancer, pancreatic cancer, liver cancer, colorectal cancer, colon cancer, gastric cancer, and small intestine cancer. In a preferred embodiment, the cancer is colorectal cancer.
In some embodiments, the dosage administered to a subject may vary with the embodiment, the drug used, the method of administration, and the site and subject to be treated. However, the dosage should be sufficient to provide a therapeutic response. A clinician may determine an effective amount to administer to a human or other subject to treat a medical condition. The precise amount required for therapeutic effectiveness may depend on a number of factors, such as the activity of the antibody and the route of administration.
The dosage of the antibodies, compositions or conjugates described herein may be administered to the mammal once or in a series of sub-doses over a suitable period of time, for example, daily, every half-week, weekly, every two weeks, every half-month, every two months, every half-year or once a year, as desired. Dosage units comprising an effective amount of the antibody, composition or conjugate may be administered in a single daily dose, or the total daily dose may be administered in two, three, four or more divided doses administered daily, as desired.
The appropriate mode of administration may be selected by the physician. The route of administration may be parenteral, for example by injection, nasal, pulmonary or transdermal. Systemic or local administration may be by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection. In some embodiments, the antibody, composition or conjugate is selected for parenteral delivery, for inhalation or for delivery through the digestive tract, e.g., oral. The administration dosage and method may vary according to the weight, age, condition, etc. of the subject, and may be appropriately selected.
In some embodiments, the method further comprises administering a second therapeutic agent to the subject. In certain embodiments, the antibodies, compositions, or conjugates disclosed herein are administered prior to, substantially simultaneously with, or after the administration of the second therapeutic agent.
In some embodiments, the second therapeutic agent is selected from the group consisting of antibodies, chemotherapeutic agents, and small molecule drugs. In some embodiments, the second therapeutic agent is selected from the group consisting of a Bruton's Tyrosine Kinase (BTK) inhibitor, PI3K inhibitor, HDAC inhibitor, ERK inhibitor, MAPK inhibitor, PD-1 inhibitor, PD-L1 inhibitor, CTLA-4 inhibitor, TIGIT inhibitor, TIM3 inhibitor, VEGF inhibitor, LAG3 inhibitor, and glucocorticoid.
In some embodiments, the second therapeutic agent is a chemotherapeutic agent. Chemotherapeutic agents may include, for example, cytotoxic agents, antimetabolites (e.g., folic acid antagonists, purine analogs, pyrimidine analogs, etc.), topoisomerase inhibitors (e.g., camptothecin derivatives, anthraquinones, anthracyclines, epipodophyllotoxins, quinoline alkaloids, etc.), antimicrotubule agents (e.g., taxanes, vinca alkaloids), protein synthesis inhibitors (e.g., cephalotaxine, camptothecin derivatives, quinoline alkaloids), alkylating agents (e.g., alkyl sulfonates, aziridines, nitrogen mustards, nitrosoureas, platinum derivatives, triazenes, etc.), alkaloids, terpenoids, and kinase inhibitors.
Medical use
The present disclosure provides the use of an antibody or antigen-binding fragment thereof disclosed herein, a bispecific antibody or antigen-binding fragment thereof disclosed herein, a pharmaceutical composition disclosed herein, or a conjugate disclosed herein in the manufacture of a medicament for treating cancer in a subject.
The disclosure also provides an antibody or antigen-binding fragment thereof disclosed herein, a bispecific antibody or antigen-binding fragment thereof disclosed herein, a pharmaceutical composition disclosed herein, or a conjugate disclosed herein for use in treating cancer in a subject.
In some embodiments of the uses disclosed herein, the cancer is a GUCY2C positive cancer. In some embodiments, the cancer is a digestive tract malignancy, preferably esophageal cancer or gastrointestinal cancer, such as colorectal cancer or gastric cancer.
In some embodiments, an antibody or antigen-binding fragment thereof disclosed herein, a bispecific antibody or antigen-binding fragment thereof disclosed herein, a pharmaceutical composition disclosed herein, or a conjugate disclosed herein is associated with a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from the group consisting of antibodies, chemotherapeutic agents, and small molecule drugs. In some embodiments, the second therapeutic agent is selected from the group consisting of a Bruton's Tyrosine Kinase (BTK) inhibitor, PI3K inhibitor, HDAC inhibitor, ERK inhibitor, MAPK inhibitor, PD-1 inhibitor, PD-L1 inhibitor, CTLA-4 inhibitor, TIGIT inhibitor, TIM3 inhibitor, VEGF inhibitor, LAG3 inhibitor, and glucocorticoid.
Diagnostic and detection methods
The present disclosure provides methods for detecting a GUCY2C protein in vitro or in vivo. In some cases, GUCY2C expression is detected in a biological sample. The sample may be any sample including, but not limited to, a blood sample, tissue from a biopsy, autopsy, and pathological specimens. Biological samples also include body fluids such as blood, serum, plasma, sputum, spinal fluid or urine. Biological samples are typically obtained from mammals, such as humans or non-human primates.
The present disclosure also provides methods of treating a subject by contacting a sample from the subject with an anti-GUCY 2C antibody disclosed herein; and detecting binding of the antibody to the sample to determine whether the subject has a GUCY2C positive cancer. An increased binding of the antibody to the sample as compared to the binding of the antibody to the control sample identifies the subject as having cancer.
In another embodiment, the present disclosure provides methods of treating a subject by contacting a sample from the subject with an anti-GUCY 2C antibody disclosed herein; and detecting binding of the antibody to the sample to diagnose a GUCY2C positive cancer in the subject. The increased binding of the antibody to the sample as compared to the binding of the antibody to the control sample confirms the diagnosis of cancer in the subject.
In some embodiments, the control sample is a sample from a subject without cancer. In particular embodiments, the sample is a blood or tissue sample.
In some embodiments of the diagnostic and detection methods, the anti-GUCY 2C antibody is directly labeled with a detectable label. In another embodiment, the anti-GUCY 2C antibody (primary antibody) is unlabeled, while the secondary antibody or other molecule that can bind to the primary antibody is labeled. As is well known to those skilled in the art, a second antibody is selected that is capable of specifically binding to a particular species and class of first antibody. For example, if the first antibody is human IgG, the second antibody may be anti-human IgG. Other molecules that can bind to antibodies include, but are not limited to, protein a and protein G, both of which are commercially available.
Suitable labels for antibodies or secondary antibodies include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, magnetic agents, and radioactive materials. Non-limiting examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase. Non-limiting examples of suitable prosthetic compoundsIllustrative examples include streptavidin/biotin and avidin/biotin. Non-limiting examples of suitable fluorescent materials include umbelliferone, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin. A non-limiting exemplary luminescent material is luminol (luminol); a non-limiting exemplary magnetic agent is gadolinium, and a non-limiting exemplary radiolabel includes 125 I、 131 I、 35 S or 3 H。
In alternative embodiments, the GUCY2C may be assayed in a biological sample by a competition immunoassay that utilizes a standard of GUCY2C protein labeled with a detectable substance and an unlabeled anti-GUCY 2C antibody. In this assay, biological samples, labeled GUCY2C protein standards, and anti-GUCY 2C antibodies are combined, and the amount of labeled GUCY2C protein standard that binds to unlabeled antibodies is determined. The amount of GUCY2C in a biological sample is inversely proportional to the amount of labeled GUCY2C protein standard that binds to anti-GUCY 2C antibodies.
The immunoassays and methods disclosed herein can be used for a variety of purposes. In one embodiment, anti-GUCY 2C antibodies can be used to detect the production of GUCY2C in cells in cell culture. In another embodiment, the antibodies can be used to detect the amount of GUCY2C in a biological sample (e.g., a tissue sample or a blood or serum sample). In some examples, the GUCY2C is a cell surface GUCY2C. In other examples, the GUCY2C protein is soluble (e.g., in a cell culture supernatant or in a bodily fluid sample such as a blood or serum sample).
Kit for detecting a substance in a sample
The present disclosure provides pharmaceutical packages or kits comprising one or more containers in which are contained one or more components of the pharmaceutical compositions described herein, such as antibodies or antigen binding fragments disclosed herein.
In particular embodiments, the kit comprises a first container comprising an antibody disclosed herein. In particular embodiments, the kit comprises a first container that is a vial containing the antibody as a lyophilized sterile powder under vacuum, and the kit further comprises a second container containing a pharmaceutically acceptable fluid.
In particular embodiments, provided herein are injection devices containing antibodies. In particular embodiments, the injection device comprises an antibody in a sterile solution. In a specific embodiment, the injection device is a syringe.
In one embodiment, the kit is provided for detecting GUCY2C in a biological sample (e.g., a blood sample or a tissue sample). For example, to confirm a cancer diagnosis in a subject, a biopsy may be performed to obtain a tissue sample for histological examination. Kits for detecting polypeptides typically comprise an anti-GUCY 2C antibody, such as any of the monoclonal antibodies disclosed herein. In further embodiments, the antibody is labeled (e.g., with fluorescence, radioactivity, or enzymatically).
In one embodiment, the kit includes instructional materials disclosing the manner of using the anti-GUCY 2C antibody. The instructional material may be written, electronic (e.g., a computer diskette or optical disk), or may be visual (e.g., a video file). The kit may also include additional components to facilitate the application for which the kit is designed. Thus, for example, the kit may additionally contain means for detecting the label (e.g., an enzyme substrate for enzymatic labeling, a filter set for detecting fluorescent labels, a suitable secondary label such as a secondary antibody, etc.). The kit may also include buffers and other reagents conventionally used to carry out particular methods. Such kits and suitable contents are well known to those skilled in the art.
In one embodiment, the diagnostic kit comprises an immunoassay. Methods for detecting GUCY2C in a biological sample typically comprise the step of contacting the biological sample with an anti-GUCY 2C antibody. Antibodies are specifically bound under immunoreaction conditions to form an immune complex, and the presence of the immune complex (bound antibody) is detected directly or indirectly.
Examples
The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the invention in any way. The present examples and methods described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Variations and other uses that fall within the spirit of the invention, as defined by the scope of the claims, will occur to those skilled in the art.
293 free style (293 FS) cells, CHO-S cells and protein A agarose were purchased from ThermoFisher Scientific. The GUCY2C positive cell line HT55 (human colon carcinoma cell line) was purchased from Cobioer Biosciences, SW948 (human colon adenocarcinoma cell line) from the national academy of sciences Shanghai cell bank (National Collection of Authenticated Cell Cultures). Human GUCY2C protein (His tag), cynomolgus monkey GUCY2C protein, mouse GUCY2C protein and human CD3 protein were purchased from ACRO. Anti-human IgG (gamma-chain specific) -R-phycoerythrin antibodies generated in goats and anti-human IgG (Fc specific) -peroxidase antibodies generated in goats were purchased from Sigma. Mouse monoclonal anti-His tag antibody (HRP) was purchased from the Sino Biological.
Two stable cell lines HT1080-GUCY2C and LS174T-GUCY2C were constructed to facilitate in vitro and in vivo efficacy studies. Briefly, a commercially available GUCY2C recombinant plasmid pCMV-GUCY2C (Sino Biological) was transiently transfected into HT1080 or LS174T cells using Lipofectamine ™ LTX reagent and PLUS ™ reagent (Thermo) and the transfection-specific medium Opti-MEM ™ I (Gibco). The cell culture was then supplemented with hygromycin B to select positive clones. After 2-3 weeks, individual positive clones were gradually isolated and validated by flow cytometry. GUCY2C positive stable cell lines HT1080-GUCY2C and LS174T-GUCY2C are obtained.
EXAMPLE 1 immunization and screening of anti-GUCY 2C antibodies
Antibodies to GUCY2C were obtained by immunizing Balb/C mice (6-8 weeks old) with recombinant human GUCY2C protein. One week after the third immunization, the titers of antisera collected from the tail vein of each mouse were determined by ELISA. Mice with the highest antiserum titers were sacrificed and spleens were removed to fuse with myeloma cells SP2/0 at a ratio of 8:1.
After 10 days of culture, obvious hybridoma cells were visible under a microscope. ELISA detects binding activity of hybridoma cell supernatants to human GUCY2C protein. ELISA was performed using standard protocols. Briefly, human GUCY2C protein was coated at 1000 ng per well on Corning EIA/RIA high binding 96 well plates (Corning Inc.), overnight at 4℃and blocked with PBS (pH 7.4) containing 3% skim milk. 30. Mu.l of hybridoma cell supernatant was added, followed by incubation at 37℃for 1 h. Each well was washed with PBS containing 0.05% Tween 20. Bound antibodies were detected by HRP conjugated goat anti-mouse IgG-Fc fragment cross-adsorbed antibodies (Bethyl). The assay was developed with TMB substrate (Solarbio) at room temperature and measured using a microplate reader at 450 nm. Binding activity of hybridoma cell supernatants to cynomolgus monkey GUCY2C protein and mouse GUCY2C protein was examined separately by using similar protocols. Hybridoma cell supernatants that bound to human and cynomolgus monkey GUCY2C proteins but not to mouse GUCY2C proteins were selected for subsequent assays.
To measure the binding capacity of hybridoma cell supernatants to cell surface GUCY2C, flow cytometry was performed using GUCY2C positive tumor cell lines HT-55 and SW 948. About 3X 10 5 Individual cells were incubated with hybridoma cell supernatants on ice for 1 h. Cells were washed once with PBS (PBSA) containing 0.5% bovine serum albumin and resuspended in 100. Mu.l of PBSA. Then 1 μl goat anti-mouse IgG (h+l) super-adsorbed secondary antibody Alexa Fluor 633 (Thermo Fisher) was added and incubated for 30 min. Cells were washed once with PBSA and used for flow cytometry analysis. Finally, a specific anti-GUCY 2C clone 56C9 was identified for use in the construction of monoclonal antibodies and bispecific antibodies.
EXAMPLE 2 construction and characterization of anti-GUCY 2C monoclonal antibodies
anti-GUCY 2C clone 56C9 was used to construct a monoclonal antibody IgG1 (designated GCC-56C9M mAb) against the intact form of human GUCY 2C. The Fab fragment of the 56C9 clone was fused to the N-terminus of the human IgG1 Fc fragment. The light chain and heavy chain were constructed separately into the vector pcDNA3.4. Construction and initial characterization of the GCC-56C9M mAb was performed as follows.
Cloning of anti-GUCY 2C monoclonal antibodies
To generate constructs for anti-GUCY 2C monoclonal antibodies, the following primers were used:
56C9-VH-F:
5 'TCCTGACTGGGTGAGGGCCGAAGTACAGTTGGTGGAGTCTGGGG 3' (sense) (SEQ ID NO: 33);
56C9-VH-R:
5'GATGGGCCCTTGGTGCTAGCTGAGGAGACGGTGACTGAGGTTCCT 3' (antisense) (SEQ ID NO: 34);
56C9-VL-F:
5'TCCTGACTGGGGTGAGGGCCCAAATTGTTCTCACCCAGTCTCCAG 3' (sense) (SEQ ID NO: 35);
56C9-VL-R:
5'GATGGTGCAGCCACCGTACGTTTCAGCTCCAGCTTGGTCCCAGTA 3' (antisense) (SEQ ID NO: 36);
Ha-F:
5 'GCTAGCACCAAGGGCCCATCGGTCTTCCCC 3' (sense) (SEQ ID NO: 37);
Lk-F:
5 'CGTACGGTGGCTGCACCATCTGTCTTCATC 3' (sense) (SEQ ID NO: 38);
pBY-vector-L/HC-RP:
5 'GGCCCTCACCCCAGTCAGGAGGAC 3' (antisense) (SEQ ID NO: 39).
A gene fragment of the VL domain of the anti-GUCY 2C antibody was amplified from anti-GUCY 2C clone 56C9 using a primer pair 56C9-VL-F/56C 9-VL-R. The gene fragment of the anti-GUCY 2C antibody VH domain was amplified from anti-GUCY 2C clone 56C9 using primer pair 56C9-VH-F/56C 9-VH-R. Fragments of the light and heavy chain vectors were cloned using primer pairs Ha-F/pBY-vector-L/HC-RP and Lk-F/pBY-vector-L/HC-RP. The gene fragments of VH and VL domains were then cloned into two light and heavy chain vectors via gibbon (Gibson) assembly.
Protein expression, purification and preliminary characterization
GCC-56C9M mAb was expressed in CHO-S cells. The plasmid and transfection agent PEI were mixed in a 1:3 ratio and then added dropwise to CHO-S cell culture broth. Cells continue to grow for 5-7 days after transfection. Cell cultures were harvested by centrifugation at 8000 rpm for 20 minutes. The culture supernatant containing the target protein was loaded onto a Protein A Sepharose Fast Flow column (GE Healthcare) and purified according to manufacturer's instructions.
Purified proteins were subjected to SDS-PAGE. On non-reducing SDS-PAGE, the GCC-56C9M mAb showed an apparent molecular weight (aMW) of about 150 kDa. On reducing SDS-PAGE, the heavy and light chains have apparent molecular weights of about 50 kDa and 25 kDa, respectively (data not shown).
The CDR sequences, light chain variable region (VL) and heavy chain variable region (VH) sequences, and the complete Light Chain (LC) and Heavy Chain (HC) sequences of the GCC-56C9M mAb according to the Kabat numbering system are shown below.
GCC-56C9M mAb LCDR1:
SASSSVSYIH (SEQ ID NO: 1)
GCC-56C9M mAb LCDR2:
STSNLAS (SEQ ID NO: 2)
GCC-56C9M mAb LCDR3:
QQRSSYPLT (SEQ ID NO: 3)
GCC-56C9M mAb HCDR1:
SYAMT (SEQ ID NO: 6)
GCC-56C9M mAb HCDR2:
TMSSGGGYTYYLDSVKG (SEQ ID NO: 7)
GCC-56C9M mAb HCDR3:
HNYGYNYAMDY (SEQ ID NO: 8)
GCC-56C9M mAb VL:
QIVLTQSPAIMSVSPGEKVTITCSASSSVSYIHWFQQRPGTSPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISRMEAEDAATYYCQQRSSYPLTFGTGTKLELK (SEQ ID NO: 4)
GCC-56C9M mAb VH:
EVQLVESGGGLVKPGGSLKLSCAASGFTFSSYAMTWVRQTPEKRLEWVATMSSGGGYTYYLDSVKGRFTISRDNAKNTLYLQMSSLRSDDTAMYYCARHNYGYNYAMDYWGQGTSVTVSS (SEQ ID NO: 9)
GCC-56C9M mAb LC:
QIVLTQSPAIMSVSPGEKVTITCSASSSVSYIHWFQQRPGTSPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISRMEAEDAATYYCQQRSSYPLTFGTGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 5)
GCC-56C9M mAb HC:
EVQLVESGGGLVKPGGSLKLSCAASGFTFSSYAMTWVRQTPEKRLEWVATMSSGGGYTYYLDSVKGRFTISRDNAKNTLYLQMSSLRSDDTAMYYCARHNYGYNYAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 10)
EXAMPLE 3 binding of anti-GUCY 2C monoclonal antibodies to GUCY2C
ELISA was performed according to standard protocols to determine the binding affinity of the GCC-56C9M mAb to human GUCY2C protein. Briefly, human GUCY2C protein was coated at 100 ng per well on Corning EIA/RIA high binding 96-well plates (Corning inc.) overnight at 4 ℃ and blocked with PBS (ph 7.4) containing 3% skim milk. Antibodies were added in five times serial dilutions from 50 μg/mL and incubated 2 h at room temperature. The plates were washed with PBS containing 1% skim milk. Bound antibody was detected by anti-Fc tag antibody (HRP) (Sigma). The assay was developed with TMB substrate (Solarbio) at room temperature and detected with a microplate reader at 450 nm. Half maximal binding (EC) was calculated by fitting the data to Langmuir adsorption isotherms 50 ). The results are shown in fig. 1.
The results showed that the GCC-56C9M mAb was tested at 193.3 ng/mL EC 50 Binding to human GUCY2C suggests that the GCC-56C9M mAb has high affinity and potentially superior properties to human GUCY 2C.
Example 4 anti-GUCY 2C monoclonal antibody mediated ADCC killing against GUCY2C positive cancer cell lines
To assess ADCC killing by the GCC-56C9M mAb, the HT1080-GUCY2C cell line was used as target cells and Jurkat-CD16a cells were used as effector cells. Target cells were treated at 1X 10 4 The density of individual cells/wells was seeded on 96-well plates and incubated overnight. The supernatant was removed and 50. Mu.l of GCC-56C9M mAb was added at a maximum concentration of 0.8. Mu.g/mL per well at 5-fold gradient. Then 1.2X10 of complete medium was incubated with 50. Mu.l of RMPI 1640 per well 5 The individual cell density was added to effector cells (Jurkat-CD 16 a). Plates were incubated in a humidified incubator at 37 ℃ for 6 hours. Then, stable-Lite luciferase assay system solution (Vazyme) was added to each well at 100. Mu.L/well, and incubated at room temperature for 10 minutes in the absence of light. Luminescence was detected using a SpectraMax 3x ELISA reader (Molecular Devices). The results are shown in fig. 2.
The results showed that the GCC-56C9M mAb exhibited concentration-dependent ADCC effect and Jurkat-CD16a cells were effectively activated by the GCC-56C9M mAb in the presence of GUCY 2C-positive HT1080-GUCY2C cells, wherein EC 50 About 24.73 ng/mL. These results indicate that ADCC is triggered by specific binding of the GCC-56C9M mAb to GUCY2C positive tumor cells HT1080-GUCY2C and recruitment of Jurkat-CD16a cells via the Fc portion of the mAb. This suggests that the GCC-56C9M mAb has the ability to induce ADCC against GUCY2C positive tumor cells.
Example 5 construction and characterization of anti-GUCY 2C bispecific antibodies
Bispecific T cell binding agents (bites) are a novel class of bispecific antibodies that direct cytotoxic T cells to kill cancer cells by binding both tumor antigens and T cell antigens such as T cell surface CD3 molecules. In this example, a specific form of BiTE, i.e., IBiTE, is designed that consists of heterodimeric light and heavy chains. The light chain comprises, from N-terminus to C-terminus, an anti-target scFv, an anti-CD 3 VL-CL and a monomeric human IgG1 Fc (mfc 7.2). The heavy chain comprises, from the N-terminus to the C-terminus, anti-CD 3 VH-CH1 and mFc7.2. Alternatively, the anti-target scFv may be fused to the N-terminus of the anti-CD 3 VH-CH 1. mfc7.2 contains two amino acid mutations (T366L and Y407H) capable of inhibiting Fc homodimerization, as described in PCT application No. PCT/US2018/016524, which is incorporated herein by reference in its entirety.
To generate GUCY2C×CD3 IBiTE, the VL and VH domains of the GCC-56C9M mAb described above were humanized to give humanized anti-GUCY 2C VL and VH domains. The humanized VL and VH domains were linked via a linker GGGGSGGGGSGGGGS (SEQ ID NO: 23) to form an anti-GUCY 2C-scFv. The scFv was fused via linker GSGGGGSGGGGS (SEQ ID NO: 24) to the N-terminus of the VL domain of the anti-CD 3 Fab. To obtain full length light chains, the anti-GUCY 2C scFv fragment was cloned by fusion cloning into a pBY plasmid containing anti-CD 3 hSP VL-CL and intact engineered Fc. The heavy chain was constructed into a single vector pBY for expression in mammalian cells. The obtained GUCY2C X CD3 IBiTE was designated GCC-56C9H4-M6MV1-1.
Two plasmids containing the heavy and light chain genes were co-transfected into 293FS or CHO-S cells. The plasmid and transfection agent PEI were mixed in a 1:3 ratio and then added dropwise to 293FS or CHO-S cell cultures. Cells continue to grow for 5-7 days after transfection. Cell cultures were harvested by centrifugation at 8000 rpm for 20 minutes. The culture supernatant containing the target protein was loaded onto a Protein A Sepharose Fast Flow column (GE Healthcare) and purified according to manufacturer's instructions.
Purified proteins were subjected to SDS-PAGE. On non-reducing SDS-PAGE, GCC-56C9H4-M6MV1-1 shows an apparent molecular weight (aMW) of about 127 kDa (data not shown).
The CDR sequences according to the Kabat numbering system, the light chain variable region (VL) and heavy chain variable region (VH) sequences, and the complete Light Chain (LC) and Heavy Chain (HC) sequences of GCC-56C9H4-M6MV1-1 are shown below.
GCC-56C9H4-M6MV1-1 LCDR1 against GUCY2C:
SASSSVSYIH (SEQ ID NO: 1)
GCC-56C9H4-M6MV1-1 LCDR2 against GUCY2C:
STSNLAS (SEQ ID NO: 2)
GCC-56C9H4-M6MV1-1 LCDR3 against GUCY2C:
QQRSSYPLT (SEQ ID NO: 3)
GCC-56C9H4-M6MV1-1 HCDR1 against GUCY2C:
SYAMT (SEQ ID NO: 6)
GCC-56C9H4-M6MV1-1 HCDR2 against GUCY2C:
TMSSGGGYTYYLDSVKG (SEQ ID NO: 7)
GCC-56C9H4-M6MV1-1 HCDR3 against GUCY2C:
HNYGYNYAMDY (SEQ ID NO: 8)
GCC-56C9H4-M6MV1-1 LCDR1 against CD3:
RSSTGAVTTSNYAN (SEQ ID NO: 13)
GCC-56C9H4-M6MV1-1 LCDR2 against CD3:
GANKRAP (SEQ ID NO: 14)
GCC-56C9H4-M6MV1-1 LCDR3 against CD3:
ALWYSNLWV (SEQ ID NO: 15)
GCC-56C9H4-M6MV1-1 HCDR1 against CD3:
TYAMN (SEQ ID NO: 17)
GCC-56C9H4-M6MV1-1 HCDR2 against CD3:
RIRSKYNNYATYYADSVKG (SEQ ID NO: 18)
GCC-56C9H4-M6MV1-1 HCDR3 against CD3:
HGNFGSSYVSYFAY (SEQ ID NO: 19)
GCC-56C9H4-M6MV1-1 VL against GUCY2C:
EIVLTQSPAIQSVSPGEKVTITCSASSSVSYIHWYQQRPGKAPKLLIYSTSNLASGVPARFSGSGSGTDFSLTISRLQAEDAATYYCQQRSSYPLTFGQGTKLEIK (SEQ ID NO: 11)
GCC-56C9H4-M6MV1-1 VH against GUCY2C:
EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMTWVRQTPEKRLEWVATMSSGGGYTYYLDSVKGRFTISRDNAKNTLYLQMSSLRADDTAVYYCARHNYGYNYAMDYWGQGTLVTVSS (SEQ ID NO: 12)
GCC-56C9H4-M6MV1-1 VL against CD3:
EIVVTQSPATLSVSPGERATLSCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGANKRAPGVPARFSGSLSGDEATLTISSLQSEDFAVYYCALWYSNLWVFGQGTKLEIK (SEQ ID NO: 16)
GCC-56C9H4-M6MV1-1 VH against CD3:
EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCARHGNFGSSYVSYFAYWGQGTTVTVSS (SEQ ID NO: 20)
GCC-56C9H4-M6MV1-1 LC:
EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMTWVRQTPEKRLEWVATMSSGGGYTYYLDSVKGRFTISRDNAKNTLYLQMSSLRADDTAVYYCARHNYGYNYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPAIQSVSPGEKVTITCSASSSVSYIHWYQQRPGKAPKLLIYSTSNLASGVPARFSGSGSGTDFSLTISRLQAEDAATYYCQQRSSYPLTFGQGTKLEIKGSGGGGSGGGGSEIVVTQSPATLSVSPGERATLSCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGANKRAPGVPARFSGSLSGDEATLTISSLQSEDFAVYYCALWYSNLWVFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGSGSCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLLCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLHSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 21)
GCC-56C9H4-M6MV1-1 HC:
EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCARHGNFGSSYVSYFAYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLLCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLHSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 22)
EXAMPLE 6 binding of GUCY2C×CD3 IBiTE to GUCY2C and CD3
To determine the binding affinity of bispecific antibody GCC-56C9H4-M6MV1-1 to human GUCY2C and human CD3, ELISA experiments were performed as described in example 3, wherein the coating protein was human GUCY2C or human CD3.
In addition, a co-binding assay was performed to further determine the binding affinity of GCC-56C9H4-M6MV 1-1. Human CD3 protein was coated at (Fc tag) 100 ng per well on Corning EIA/RIA high binding 96-well plates (Corning inc.) overnight at 4 ℃ and blocked with PBS (ph 7.4) containing 3% skim milk. Human GUCY2C protein 100 ng per well and 5-fold serial dilutions of antibody from 10 μg/mL were then added simultaneously and incubated at room temperature for 2 h. The plates were washed with PBS containing 1% skim milk. The bound antibody was detected by anti-His tag antibody (HRP) (Sino Biological). The assay was developed with TMB substrate (Solarbio) at room temperature and detected with a microplate reader at 450 nm. Half maximal binding (EC) was calculated by fitting the data to Langmuir adsorption isotherms 50 ). The results are shown in FIGS. 3A-3C.
The results showed that GCC-56C9H4-M6MV1-1 had an EC of 139.9 ng/mL 50 In combination with human GUCY2C (FIG. 3A), EC at 115.6 ng/mL 50 Bind human CD3 (fig. 3B). Furthermore, the results of the co-binding analysis showed that GCC-56C9H4-M6MV1-1 had an EC of 279.7 ng/mL 50 Simultaneously binding to both the GUCY2C and CD3 proteins (fig. 3C). These results indicate that GCC-56C9H4-M6MV1-1 is capable of binding GUCY2C and CD3 with high affinity, suggesting that GCC-56C9H4-M6MV1-1 is potentially highly potentIs an anti-tumor activity of (a).
EXAMPLE 7 binding of GUCY2C×CD3 IBiTE to cancer cell lines
To measure the binding capacity of GUCY2C×CD3 IBite to cell surface expressed GUCY2C, flow cytometry was performed using the GUCY2C positive cell lines HT55, SW948 and LS174T-GUCY 2C. Will be about 5 x 10 5 Individual cells were incubated with different concentrations of antibody (50, 10, 2, 0.4, 0.08, 0.016, 0 ng/mL) for 1h on ice. Cells were washed once with PBS (PBSA) containing 0.5% bovine serum albumin and resuspended in 100 μl PBSA. Then 1 μl/test anti-human IgG (gamma chain specific) -R-phycoerythrin antibody was added and incubated for 30 min. Cells were washed once with PBSA and then used for flow cytometry analysis. The results are shown in FIGS. 4A-4C.
The results showed that GCC-56C9H4-M6MV1-1 bound well to the GUCY2C positive cell lines HT55, SW948 and LS174T-GUCY2C in a concentration-dependent manner.
Example 8 GUCY2C×CD3IBiTE mediated T cell activation
GUCY2C X CD3 IBiTE's ability and specificity to activate human T cells in the presence of target cells (GUCY 2C positive cells HT55, SW948, LS174T-GUCY 2C) by using Bio-Glo TM Luciferase assay system and TCR/CD3 effector cells (Jurkat-NFAT-CD 3). TCR/CD3 effector cells (Jurkat-NFAT-CD 3) express endogenous TCR and CD3 receptors. When effector cells (Jurkat-NFAT-CD 3) are conjugated to appropriate TCR/CD3 ligands or anti-TCR/CD 3 antibodies, the TCR transduces intracellular signals, resulting in TCR-mediated T cell activation and the generation of an enhanced fluorescent signal.
Target cells were grown in 100. Mu.L of RMPI 1640 complete medium at 6X 10 per well 4 (for HT55 and LS174T-GUCY 2C) or 3X 10 4 The density of the cells (for SW 948) was seeded overnight in 96-well plates. The supernatant was removed and 50. Mu.l of GCC-56C9H4-M6MV1-1 diluted 5-fold in a gradient was added per well at a maximum concentration of 100. Mu.g/mL. Then 1.5X10 total medium at 50. Mu.l RMPI 1640 per well 5 Density of individual cells effector cells (Jurkat-NFAT-CD 3) were added. Plates were incubated in a humidified incubator at 37 ℃ for 6 hours. Then, stable-Lite luciferase assay system solution (Vazy me) was added to each well at 100 μl/well and incubated at room temperature for 10 min protected from light. Luminescence was detected using a SpectraMax 3x ELISA reader (Molecular Devices). The results are shown in FIGS. 5A-5C.
Effector cells (Jurkat-NFAT-CD 3) are activated efficiently by GCC-56C9H4-M6MV1-1, EC in the presence of GUCY2C positive HT55, SW948 and LS174T-GUCY2C cells 50 About 374.1 ng/mL, 518.9 ng/mL and 334.6 ng/mL, respectively. These results indicate that GCC-56C9H4-M6MV1-1 can bind both the CD3 antigen of effector cells and the GUCY2C antigen of tumor cells, resulting in T cell specific activation.
EXAMPLE 9 GUCY2C×CD3IBiTE mediated killing of human cancer cell lines
Bispecific T cell cements can bind both tumor antigens and T cell antigens (e.g., CD3 molecules on the surface of T cells), resulting in T cell aggregation and activation, ultimately leading to killing of tumor cells. To evaluate the killing efficiency of GUCY2C×CD3 IBiTE, the GUCY2C positive tumor cell lines HT55, SW948 and LS174T-GUCY2C were used as target cells.
Target cells were plated in 100. Mu.L per well of RMPI 1640 complete medium at 4X 10 4 (for HT55 and LS174T-GUCY 2C) or 1.3X10 4 The density of (for SW 948) cells was seeded on 96-well plates and incubated for about 20h. Then 1X 10 in complete medium at 50. Mu.L per well of RMPI 1640 5 Individual cell density effector cell human PBMC (hPBMC) was added. At the same time, 50 μl of antibody diluted five times from 8 μg/ml was added to each well and incubated for 48 h (for HT55 and LS174T-GUCY 2C) or 24h (for SW 948). After incubation, the medium was removed from the target cells and 100 μl of RPMI 1640 complete medium containing 10% CCK8 was added to the medium at CO 2 Incubate in incubator for 30 min. Cell killing activity was measured using an enzyme-labeled instrument according to the manufacturer's instructions. The results are shown in FIGS. 6A-6C.
The results show that GCC-56C9H4-M6MV1-1 has effective killing effect on HT55, SW948 and LS174T-GUCY2C cells. The EC50 of GCC-56C9H4-M6MV1-1 killed HT55 cells was 15.24 ng/mL (FIG. 6A), the EC50 of GCC-56C9H4-M6MV1-1 killed SW948 cells was 12.02 ng/mL (FIG. 6B), and the EC50 of GCC-56C9H4-M6MV1-1 killed LS174T-GUCY2C cells was 2.74 ng/mL (FIG. 6C). These results indicate that GCC-56C9H4-M6MV1-1 has effective killing ability on GUCY2C positive tumor cell lines HT55, SW948 and LS174T-GUCY2C, and indicate that GCC-56C9H4-M6MV1-1 has remarkable in vitro anti-tumor efficacy.
Example 10 tumor growth inhibition in GUCY2C×CD3IBiTE mediated mice
Will be 2X 10 6 HT1080-GUCY2C cells (100. Mu.L) and 1.7X10 6 A total of 200. Mu.L of the mixture of hBMCs (100. Mu.L) was inoculated subcutaneously into the right flank of B-NDG mice and the mice were randomly divided into three groups (five mice per group). The experimental groups were low dose (100. Mu.g/kg of GCC-56C9H4-M6MV1-1 by intravenous injection) and high dose (500. Mu.g/kg of GCC-56C9H4-M6MV1-1 by intravenous injection) and the negative control group was treated with PBS. Mice received 3 treatments per week for a total of 9 days. At the same time, tumor volume and mouse body weight were measured for 9 days. Tumor Growth Inhibition (TGI) after 9 days of treatment was calculated using the following formula: TGI (%) = (C-T)/c×100 (T: mean tumor volume of experimental group; C: mean tumor volume of control group). The results are shown in FIGS. 7A-7B.
The results showed that GCC-56C9H4-M6MV1-1 showed effective inhibition of tumor growth, and tumors disappeared after 3 administrations in both the high dose group (500. Mu.g/kg) and the low dose group (100. Mu.g/kg) (FIG. 7A). The body weight of all mice groups was only slightly changed (FIG. 7B), indicating the low toxicity advantage of GCC-56C9H4-M6MV1-1 in vivo.
In summary, in vivo studies have shown that GCC-56C9H4-M6MV1-1 can specifically and effectively inhibit the growth of GUCY2C positive tumor cells. Therefore, GCC-56C9H4-M6MV1-1 is expected to develop clinical studies.
While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Many modifications, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments described herein may be employed. The following claims are intended to define the scope of the invention and to cover methods and structures within the scope of these claims and their equivalents.
Claims (34)
1. An antibody or antigen-binding fragment thereof that specifically binds to GUCY2C, said antibody or antigen-binding fragment thereof comprising a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL comprises an amino acid sequence set forth in SEQ ID NO:1-3, and said VH comprises amino acid sequences set forth in SEQ ID NOs: 6-8, HCDR 1-3.
2. The antibody or antigen-binding fragment thereof of claim 1, wherein
(i) The VL comprises a sequence identical to SEQ ID NO:4, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:9 has an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity; or alternatively
(ii) The VL comprises a sequence identical to SEQ ID NO:11, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:12, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity.
3. The antibody or antigen-binding fragment thereof of claim 2, wherein
(i) The VL comprises an amino acid sequence as set forth in SEQ ID NO. 4 and the VH comprises an amino acid sequence as set forth in SEQ ID NO. 9; or alternatively
(ii) The VL comprises the amino acid sequence shown as SEQ ID NO. 11 and the VH comprises the amino acid sequence shown as SEQ ID NO. 12.
4. The antibody or antigen-binding fragment thereof of any one of claims 1-3, wherein the antibody is a murine antibody, a chimeric antibody, a humanized antibody, or a human antibody.
5. The antibody or antigen binding fragment thereof of any one of claims 1-3, wherein the antibody belongs to an isotype selected from IgG, igA, igM, igE and IgD.
6. The antibody or antigen binding fragment thereof of any one of claims 1-3, wherein the antibody belongs to a subtype selected from the group consisting of IgG1, igG2, igG3, and IgG 4.
7. The antibody or antigen-binding fragment thereof of any one of claims 1-3, wherein the antigen-binding fragment is selected from the group consisting of Fab, fab ', F (ab') 2 Fv and scFv.
8. The antibody or antigen-binding fragment thereof of any one of claims 1-3, wherein the antigen-binding fragment is a ds-scFv.
9. The antibody or antigen-binding fragment thereof of any one of claims 1-3, wherein the antibody is a monoclonal antibody.
10. The antibody or antigen-binding fragment thereof of claim 9, wherein the antibody comprises a light chain comprising a sequence identical to SEQ ID NO:5, and the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:10, an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity.
11. A bispecific antibody or antigen-binding fragment thereof comprising a first antigen-binding region that binds to GUCY2C comprising a first light chain variable region (VL 1) and a first heavy chain variable region (VH 1) and a second antigen-binding region that binds to CD3 comprising a second light chain variable region (VL 2) and a second heavy chain variable region (VH 2), wherein
The VL1 comprises amino acid sequences shown in SEQ ID NO:1-3, and said VH1 comprises amino acid sequences set forth in SEQ ID NOs: 6-8 HCDR 1-3; and
the VL2 comprises amino acid sequences shown in SEQ ID NO:13-15, and said VH2 comprises amino acid sequences set forth in SEQ ID NOs: 17-19, HCDR 1-3.
12. The bispecific antibody or antigen-binding fragment thereof of claim 11, wherein
(i) The VL1 comprises a sequence identical to SEQ ID NO:4 and said VH1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:9 has an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity; or alternatively
(ii) The VL1 comprises a sequence identical to SEQ ID NO:11 and said VH1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:12, an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity; and wherein
The VL2 comprises a sequence identical to SEQ ID NO:16 and said VH2 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:20, an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity.
13. The bispecific antibody or antigen-binding fragment thereof of claim 12, wherein
(i) The VL1 comprises an amino acid sequence as shown in SEQ ID NO. 4 and the VH1 comprises an amino acid sequence as shown in SEQ ID NO. 9; or (b)
(ii) The VL1 comprises an amino acid sequence as shown in SEQ ID NO. 11 and the VH1 comprises an amino acid sequence as shown in SEQ ID NO. 12; and is also provided with
The VL2 comprises an amino acid sequence as shown in SEQ ID NO. 16 and the VH2 comprises an amino acid sequence as shown in SEQ ID NO. 20.
14. The bispecific antibody or antigen-binding fragment thereof of any one of claims 11-13, wherein the first antigen-binding region comprises an scFv comprising the VL1 and the VH1, and the scFv is linked to the VL2 or the N-terminus of VH2, optionally via a linker.
15. The bispecific antibody or antigen-binding fragment thereof of claim 14, wherein the bispecific antibody comprises:
a first polypeptide chain comprising, from N-terminus to C-terminus: the scFv, the optional linker, the VL2, the light chain constant region (CL), the heavy chain constant region 2 (CH 2), and the heavy chain constant region 3 (CH 3); and
a second polypeptide chain comprising, from N-terminus to C-terminus: the VH2, heavy chain constant region 1 (CH 1), heavy chain constant region 2 (CH 2), and heavy chain constant region 3 (CH 3).
16. The bispecific antibody or antigen-binding fragment thereof of claim 14, wherein the linker comprises an amino acid sequence selected from (G4S) n and GS (G4S) n, wherein n is an integer selected from 1-5.
17. The bispecific antibody or antigen-binding fragment thereof of claim 16, wherein the linker comprises the amino acid sequence as set forth in SEQ ID NO:23 or 24.
18. The bispecific antibody or antigen-binding fragment thereof of claim 15, wherein the first polypeptide chain comprises a sequence that hybridizes to SEQ ID NO:21, and the second polypeptide chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:22 has an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity.
19. The bispecific antibody or antigen-binding fragment thereof of any one of claims 11-13, wherein the bispecific antibody is a bispecific T cell cement (BiTE).
20. A nucleic acid comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof according to any one of claims 1-10 or the bispecific antibody or antigen-binding fragment thereof according to any one of claims 11-19.
21. A vector comprising the nucleic acid of claim 20.
22. A host cell comprising the nucleic acid of claim 20 or the vector of claim 21.
23. A pharmaceutical composition comprising (i) an antibody or antigen-binding fragment thereof according to any one of claims 1-10, or a bispecific antibody or antigen-binding fragment thereof according to any one of claims 11-19; and (ii) a pharmaceutically acceptable carrier or excipient.
24. The pharmaceutical composition of claim 23, further comprising a second therapeutic agent.
25. The pharmaceutical composition of claim 24, wherein the second therapeutic agent is selected from the group consisting of an antibody, a chemotherapeutic agent, and a small molecule drug.
26. The pharmaceutical composition of claim 24 or 25, wherein the second therapeutic agent is selected from the group consisting of a Bruton's Tyrosine Kinase (BTK) inhibitor, PI3K inhibitor, HDAC inhibitor, ERK inhibitor, MAPK inhibitor, PD-1 inhibitor, PD-L1 inhibitor, CTLA-4 inhibitor, TIGIT inhibitor, TIM3 inhibitor, VEGF inhibitor, LAG3 inhibitor, and glucocorticoid.
27. A conjugate comprising an antibody or antigen-binding fragment thereof according to any one of claims 1-10, or a bispecific antibody or antigen-binding fragment thereof according to any one of claims 11-19, and a chemical moiety conjugated thereto.
28. The conjugate of claim 27, wherein the chemical moiety is selected from the group consisting of a therapeutic agent, a detectable moiety, and an immunostimulatory molecule.
29. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-10, the bispecific antibody or antigen-binding fragment thereof according to any one of claims 11-19, the pharmaceutical composition according to any one of claims 23-26, or the conjugate according to claim 27 or 28 in the manufacture of a medicament for treating cancer in a subject, wherein the cancer is a GUCY2C positive cancer and the cancer is a gastrointestinal cancer.
30. The use of claim 29, wherein the cancer is esophageal cancer.
31. The use of claim 29, wherein the cancer is colorectal or gastric cancer.
32. The use of any one of claims 29-31, wherein the medicament is in combination with a second therapeutic agent.
33. The use of claim 32, wherein the second therapeutic agent is selected from the group consisting of an antibody, a chemotherapeutic agent, and a small molecule drug.
34. The use of claim 33, wherein the second therapeutic agent is selected from the group consisting of a Bruton's Tyrosine Kinase (BTK) inhibitor, PI3K inhibitor, HDAC inhibitor, ERK inhibitor, MAPK inhibitor, PD-1 inhibitor, PD-L1 inhibitor, CTLA-4 inhibitor, TIGIT inhibitor, TIM3 inhibitor, VEGF inhibitor, LAG3 inhibitor, and glucocorticoid.
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