Methods of treating cancer using anti-CD22 antibody oligonucleotide conjugates

Abstract

The present disclosure provides a method of treating cancer using an anti-CD22 antibody-oligonucleotide conjugate.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 343,506, filed on May 18, 2022, which is incorporated herein by reference in its entirety.

[0003] Reference to an electronic sequence listing

[0004] The contents of the electronic sequence listing (186492001040seqlist.xml; size: 319,072 bytes; and creation date: May 3, 2023) are incorporated herein by reference in their entirety. Technical Field

[0005] The present disclosure generally relates to methods of treating cancer using anti-CD22 antibody-oligonucleotide conjugates. Background Art

[0006] Immune-mediated cancer elimination requires the coordination of the innate (e.g., dendritic cells, macrophages, myeloid-derived suppressor cells) and adaptive (i.e., B cells, cytotoxic T cells, regulatory T cells) immune systems. Current gold standard cancer treatments involve the administration of immune checkpoint blockade therapies (anti-PD-1, anti-CTLA-4), but these therapies are only effective in up to 30% of patients and can have severe or even fatal side effects in 10-30% of cancer patients. In addition, these therapies primarily increase T lymphocyte activity exclusively, thereby ignoring the importance of a coordinated immune response. Therefore, there remains a need for a safe and effective treatment that exploits a broad range of possible immune engagements to eliminate cancer. Summary of the invention

[0007] In one aspect, the present invention provides a method for treating cancer in an individual, comprising administering to the individual a conjugate comprising an anti-CD22 antibody (Ab) and an immunomodulatory oligonucleotide (P) at a dose of 0.1 mg / kg to 60 mg / kg; wherein Ab comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides (Q); wherein each of the two Q tag peptides comprises the amino acid sequence RPQGFGPP (SEQ ID NO: 49); wherein one of the Q tag peptides is connected to the C-terminus of each of the two antibody heavy chains; wherein one of the two Q tag peptides is connected to the immunomodulatory oligonucleotide via an amide bond to a glutamine residue of the Q tag peptide and a linker (L), as shown in formula (A):

[0008]

[0009] in represents the connection point of Q to antibody (Ab); wherein each heavy chain of Ab comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 113, a CDR2 comprising an amino acid sequence of SEQ ID NO: 115, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 116, and each light chain of Ab comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 117, a CDR2 comprising an amino acid sequence of SEQ ID NO: 119, and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 120-122; wherein linker L is Where m is 24, and where represents the point of attachment to oligonucleotide P, and represents the point of attachment to the glutamine residue of the Q tag; and wherein the oligonucleotide P comprises the following structure:

[0010] or

[0011] or a pharmaceutically acceptable salt thereof,

[0012] in and represents the attachment point within the oligonucleotide, where Indicates the connection point with the linker L.

[0013] In some embodiments, the individual has an inoperable, locally advanced, metastatic, and / or recurrent solid tumor. In some embodiments, the individual has a cancer selected from the group consisting of: cutaneous melanoma, breast cancer, ovarian cancer, colorectal cancer, non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC), head and neck squamous cell carcinoma (HNSCC), Merkel cell carcinoma, hepatocellular carcinoma (HCC), cervical squamous cell carcinoma, urothelial carcinoma, endometrial carcinoma, gastroesophageal adenocarcinoma, and bile duct cancer.

[0014] On the other hand, provided herein is a method for treating cancer in an individual, comprising administering to the individual a conjugate comprising an anti-CD22 antibody (Ab) and an immunomodulatory oligonucleotide (P); wherein the individual suffers from an inoperable, locally advanced, metastatic and / or recurrent solid tumor; wherein Ab comprises two antibody light chains, two antibody heavy chains and two Q tag peptides (Q); wherein each of the two Q tag peptides comprises the amino acid sequence RPQGFGPP (SEQ ID NO: 49); wherein one of the Q tag peptides is linked to the C-terminus of each of the two antibody heavy chains; wherein one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond to a glutamine residue of the Q tag peptide and a linker (L), as shown in formula (A):

[0015]

[0016] in represents the connection point of Q to antibody (Ab); wherein each heavy chain of Ab comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 113, a CDR2 comprising an amino acid sequence of SEQ ID NO: 115, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 116, and each light chain of Ab comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 117, a CDR2 comprising an amino acid sequence of SEQ ID NO: 119, and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 120-122; wherein linker L is Where m is 24, and where represents the point of attachment to oligonucleotide P, and represents the point of attachment to the glutamine residue of the Q tag; and wherein the oligonucleotide P comprises the following structure:

[0017] or

[0018] or a pharmaceutically acceptable salt thereof,

[0019] in and represents the point of attachment within the oligonucleotide, and wherein Indicates the connection point with the linker L.

[0020] In some embodiments, the individual has a cancer selected from the group consisting of: cutaneous melanoma, breast cancer, ovarian cancer, colorectal cancer, non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC), head and neck squamous cell carcinoma (HNSCC), Merkel cell carcinoma, hepatocellular carcinoma (HCC), cervical squamous cell carcinoma, urothelial carcinoma, endometrial carcinoma, gastroesophageal adenocarcinoma, and bile duct cancer. In some embodiments, the conjugate is administered to the individual at a dose of between 0.1 mg / kg and 60 mg / kg.

[0021] In some embodiments according to any embodiment described herein, the individual suffers from a solid tumor that progresses under standard therapy and / or is intolerant to standard therapy. In some embodiments, the conjugate is administered to an individual at a dose of 0.1, 0.3, 1.0, 3.0, 10.0, 30.0 or 60.0 mg / kg. In some embodiments, the conjugate is administered to an individual once every two weeks. In some embodiments, each heavy chain of Ab comprises a CDR1 containing an amino acid sequence of SEQ ID NO: 113, a CDR2 containing an amino acid sequence of SEQ ID NO: 115, and a CDR3 containing an amino acid sequence of SEQ ID NO: 116, and each light chain of Ab comprises a CDR1 containing an amino acid sequence of SEQ ID NO: 117, a CDR2 containing an amino acid sequence of SEQ ID NO: 119, and a CDR3 containing an amino acid sequence of SEQ ID NO: 120. In some embodiments, each heavy chain of Ab comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 113, a CDR2 comprising an amino acid sequence of SEQ ID NO: 115, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 116, and each light chain of Ab comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 117, a CDR2 comprising an amino acid sequence of SEQ ID NO: 119, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 121. In some embodiments, each heavy chain of Ab comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 113, a CDR2 comprising an amino acid sequence of SEQ ID NO: 115, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 116, and each light chain of Ab comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 117, a CDR2 comprising an amino acid sequence of SEQ ID NO: 119, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 122. In some embodiments, each heavy chain of Ab comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 65. In some embodiments, each light chain of Ab comprises a light chain variable region comprising an amino acid sequence of SEQ ID NO: 73. In some embodiments, each light chain of Ab comprises a light chain variable region comprising an amino acid sequence of SEQ ID NO: 87. In some embodiments, each heavy chain of Ab comprising a Q tag peptide comprises an amino acid sequence of SEQ ID NO: 179 or 180; and wherein each light chain of Ab comprises an amino acid sequence of SEQ ID NO: 181 or 182. In some embodiments, each heavy chain of Ab comprising a Q tag peptide comprises an amino acid sequence of SEQ ID NO: 179; and wherein each light chain of Ab comprises an amino acid sequence of SEQ ID NO: 181.In some embodiments, each heavy chain of the Ab comprising a Q tag peptide comprises an amino acid sequence of SEQ ID NO: 179; and wherein each light chain of the Ab comprises an amino acid sequence of SEQ ID NO: 182. In some embodiments, each heavy chain of the Ab comprising a Q tag peptide comprises an amino acid sequence of SEQ ID NO: 180; and wherein each light chain of the Ab comprises an amino acid sequence of SEQ ID NO: 181. In some embodiments, each heavy chain of the Ab comprising a Q tag peptide comprises an amino acid sequence of SEQ ID NO: 180; and wherein each light chain of the Ab comprises an amino acid sequence of SEQ ID NO: 182. In some embodiments, the individual is a human. In some embodiments, the conjugate is administered in the form of a formulation comprising a conjugate at a concentration of 30 mg / mL, 20 mM citrate, 150 mM L-arginine, 50 mM NaCl, and 0.02% polysorbate 80 (w / v); wherein the pH of the formulation is 6.5. In some embodiments, the conjugate is administered intravenously to the individual. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The present application may be understood by referring to the following description in conjunction with the accompanying drawings.

[0023] Figure 1 A schematic diagram of an exemplary antibody:CpG conjugate is shown in which an engineered Q tag (RPQGFGPP; SEQ ID NO:49) is fused to the C-terminus of each heavy chain (with DAR 1).

[0024] Figure 2A Shown are the expression levels of cell surface CD22 on peripheral B cells of patients after treatment with anti-CD22-CpG conjugates.

[0025] Figure 2B Shown are cell surface CD22 expression levels on peripheral B cells of patients on day 1 before treatment with the indicated doses of anti-CD22-CpG conjugate (C1D1 pre), 3 hours on day 1 (3 hours on the far left), day 2 (C1D2), day 8 (C1D8), 15 hours before administration of the second dose (i.e., second cycle; C1D15 pre), 3 hours after administration of the second dose (3 hours on the far right), day 16 during the second cycle (C2D16), and day 22 during the second cycle (C2D22).

[0026] Figure 3A Shown is the expression of CD86 on naive B cells following treatment with anti-CD22-CpG conjugates at the indicated doses in patients.

[0027] Figure 3BShown is the expression of CD86 on memory B cells following treatment with anti-CD22-CpG conjugates at the indicated doses in patients.

[0028] Figure 3C Shown is the expression of CD86 on naive B cells following treatment with anti-CD22-CpG conjugates in patients at the indicated doses and time points.

[0029] Figure 4 Shown is the concentration of anti-CD22-CpG conjugate (TRAAC) in patients over time following administration of 0.1 mg / kg, 0.3 mg / kg or 1 mg / kg of the conjugate. DETAILED DESCRIPTION

[0030] The following description sets forth exemplary methods, parameters, etc. However, it should be appreciated that such description is not intended to limit the scope of the present disclosure, but is provided as a description of exemplary embodiments.

[0031] Provided herein are methods for treating solid tumors, comprising administering a conjugate comprising an anti-CD22 antibody and an immunomodulatory oligonucleotide. In some embodiments, the cancer is an advanced or metastatic solid tumor. In some embodiments, the conjugate is administered to an individual at a dose of 0.1 mg / kg to 60 mg / kg.

[0032] Toll-like receptor 9 (TLR9), also known as CD289, is a transmembrane and intracellular pattern recognition receptor that recognizes pathogen-associated molecular patterns (PAMPs) to participate in immune responses in the absence of pathogens. TLR9 is an important receptor expressed by immune cells including dendritic cells (DCs), B lymphocytes, macrophages, natural killer cells, and other antigen-presenting cells. TLR9 activation triggers intracellular signaling cascades, leading to the activation, maturation, proliferation, and cytokine production of these immune cells, thereby bridging and coordinating innate and adaptive immunity. Martinez-Campos et al., Viral Immunol. 2016, 30, 98-105; Notley et al., Sci. Rep. 2017, 7, 42204. Therefore, identifying new TLR9 agonist compounds can activate innate and adaptive immunity and be used as potential anticancer therapeutics.

[0033] Natural TLR9 agonists include oligodeoxynucleotides (CpG ODN) containing unmethylated cytosine-guanine dinucleotides (CpG). CpG ODN is usually easily degraded in serum, so the pharmacokinetics of CpG ODN may be one of the limiting factors for its development as a therapeutic agent. In addition, CpG ODN exhibits uneven tissue distribution, which can lead to increased PAMP-related toxicity. These shortcomings can be overcome by conjugating CpG ODN with a targeting moiety for treating a specific disease, such as an anti-CD22 antibody for anticancer therapy. In fact, CD22 is a transmembrane sialic acid glycoprotein receptor, and its expression on B cells including tumor-infiltrating B cells (TIB) is limited, which will be internalized with anti-CD22-CpG conjugates to deliver TLR9 agonists to stimulate anti-cancer immune responses. Preclinical mouse tumor models show that anti-CD22-CpG conjugates activate TLR9 pathways in B cells to increase immune activation, cytokine production, antigen presentation, and anti-tumor efficacy. However, there is still a need to evaluate the safety and efficacy of such drug compounds in human cancer patients.

[0034] I. Definitions

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. All patents, applications, published applications, and other publications mentioned herein are incorporated herein by reference in their entirety. If the definitions set forth in this section are contrary to or otherwise inconsistent with the definitions set forth in the patents, applications, or other publications incorporated herein by reference, the definitions set forth in this section take precedence over the definitions incorporated herein by reference.

[0036] It should be understood that certain features of the disclosure described in the context of separate embodiments for the sake of clarity may also be provided in combination in a single embodiment. Conversely, various features of the disclosure described in the context of a single embodiment for the sake of brevity may also be provided separately or in any suitable sub-combination. All combinations of embodiments with respect to specific method steps, reagents, or conditions are specifically encompassed by the disclosure and are disclosed herein as if each combination were individually and explicitly disclosed.

[0037] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should also be noted that the claims may be drafted to exclude any optional elements. Thus, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "only," "only" and the like in connection with claim element recitations, or use of a "negative" limitation.

[0038] In this application, unless the context indicates otherwise, references to compounds of formula (A) include ionic forms, polymorphs, pseudopolymorphs, amorphous forms, solvates, co-crystals, chelates, isomers, tautomers, oxides (e.g., N-oxides, S-oxides), esters, prodrugs, isotopes, and / or protected forms thereof. In some embodiments, references to compounds of formula (A) include polymorphs, solvates, co-crystals, isomers, tautomers, and / or oxides thereof. In some embodiments, references to compounds of formula (A) include polymorphs, solvates, and / or co-crystals thereof. In some embodiments, references to compounds of formula (A) include isomers, tautomers, and / or oxides thereof. In some embodiments, references to compounds of formula (A) include solvates thereof.

[0039] The term "immunomodulatory polynucleotide" or "immunomodulatory oligonucleotide" as used herein represents a polynucleotide construct containing a total of 6 to 50 consecutive nucleosides. The immunomodulatory polynucleotide is capable of regulating the innate immune response, which is determined by changes in activation in intracellular signaling pathways, including but not limited to NFκB, changes in activation marker expression, or changes in the secretion of at least one inflammatory cytokine or at least one type I interferon in immune cells (e.g., antigen presenting cells) to which the immunomodulatory polynucleotide is delivered (e.g., compared to another immune cell (e.g., antigen presenting cell) to which the immunomodulatory polynucleotide is delivered) or immune cells (e.g., antigen presenting cells) interacting with the immune cells to which the immunomodulatory polynucleotide is delivered (including direct cell-to-cell interactions and indirect stimulation, such as one or more cytokines secreted from cells to which the immunomodulatory polynucleotide is delivered).

[0040] As used herein, the term "immunostimulatory polynucleotide" or "immunostimulatory oligonucleotide" refers to an immunomodulatory polynucleotide that is capable of activating an immune response, such as by, for example, increased activation of intracellular signaling pathways such as NFκB or increased levels of cell surface activation or functional markers, or increased secretion of at least one inflammatory cytokine or at least one type I interferon in an immune cell (e.g., an antigen presenting cell) (e.g., compared to another immune cell (e.g., an antigen presenting cell) to which the immunostimulatory polynucleotide is not delivered), or in an immune cell that interacts with the immune cell (e.g., an antigen presenting cell) to which the immunomodulatory polynucleotide is delivered (including direct cell-to-cell interactions as well as indirect stimulation, for example, secretion of one or more cytokines from the cell to which the immunomodulatory polynucleotide is delivered).

[0041] It should be understood that the terms "polynucleotide" and "oligonucleotide" are used interchangeably herein. It should also be understood that the terms "immunomodulatory polynucleotide", "immunostimulatory polynucleotide", "immunosuppressive polynucleotide" and "conjugate" encompass immunomodulatory polynucleotides, immunostimulatory polynucleotides, immunosuppressive polynucleotides and salts of conjugates, respectively. For example, the terms "immunomodulatory polynucleotide", "immunostimulatory polynucleotide", "immunosuppressive polynucleotide" and "conjugate" encompass protonated neutral forms of phosphates, phosphorothioates or phosphorodithioates (P-XH moieties, wherein X is O or S) and deprotonated ionic forms of phosphates, phosphorothioates or phosphorodithioates (PX - moiety, wherein X is O or S). Therefore, it should be understood that the E1 , R E2 and R E3 Phosphate and phosphodiester of one or more hydrogen atoms in the polynucleotide encompass salts, wherein phosphate, phosphorothioate or phosphorodithioate exist in the form of deprotonated ions. In addition, the terms "free", "naked" and "unbound" referring to immunomodulatory polynucleotides, immunostimulatory polynucleotides, immunosuppressive polynucleotides and / or oligonucleotides (e.g., CpG oligonucleotides) are used interchangeably herein.

[0042] The term "phosphotriester" as used herein refers to a phosphate ester in which all three valences are replaced by non-hydrogen substituents. A phosphotriester is composed of: a phosphate, a phosphorothioate, or a phosphorodithioate; one or two bonds to a nucleoside, an abasic spacer, and / or a phosphoryl group; and one or two groups independently selected from the group consisting of a bioreversible group, a non-bioreversible group, an auxiliary part, a conjugated group, and a linker that is bound to a targeting moiety and optionally to one or more (e.g., 1 to 6) auxiliary parts. A terminal phosphotriester includes a bond connected to a nucleoside-containing group and two groups independently selected from the group consisting of: a bioreversible group; a non-bioreversible group; an auxiliary part; a conjugated group; a phosphoryl group; and a linker that is bound to a targeting moiety and optionally to one or more (e.g., 1 to 6) auxiliary parts. In some embodiments, a terminal phosphotriester contains 1 or 0 linkers that are bound to a targeting moiety and optionally to one or more (e.g., 1 to 6) auxiliary parts. An internucleoside phosphotriester comprises two bonds connected to a nucleoside-containing group. The phosphate triester can be a group of the following structures:

[0043]

[0044] in:

[0045] X E1 and X E2 Each independently is O or S;

[0046] R E1 and R E3 Each is independently a bond to a nucleoside; a sugar analog without a basic spacer; a bioreversible group; a non-bioreversible group; an auxiliary part; a conjugated group; a linker bound to a targeting part; a linker bound to a targeting part and one or more (e.g., 1 to 6) auxiliary parts; or a compound of the formula -P(=X E1 )(–X E2 –R E2 A) the phosphorus atom in the –O– group,

[0047] Where R E2 A is hydrogen; a bioreversible group; a non-bioreversible group; an auxiliary moiety; a conjugating group; a linker bound to a targeting moiety; or a linker bound to a targeting moiety and one or more (e.g., 1 to 6) auxiliary moieties; and

[0048] R E2 is a bioreversible group; a non-bioreversible group; an auxiliary moiety; a conjugated group; a linker bound to a targeting moiety; or a linker bound to a targeting moiety and one or more (e.g., 1 to 6) auxiliary moieties;

[0049] The condition is R E1 and R E3 At least one of the bonds is to a group comprising at least one nucleoside.

[0050] If R E1 and R E3 If R is bound to a group containing at least one nucleoside, then the phosphotriester is an internucleoside phosphotriester. E1 and R E3 If one and only one of the bonds is connected to the group containing a nucleoside, the phosphotriester is a terminal phosphotriester.

[0051] The term "amino acid" as used herein refers to any amino acid (standard and non-standard amino acids), including but not limited to α-amino acids, β-amino acids, γ-amino acids and δ-amino acids. Examples of suitable amino acids include but are not limited to alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, serine, tyrosine, arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine. Other examples of suitable amino acids include but are not limited to ornithine, hypusine, 2-aminoisobutyric acid, dehydroalanine, γ-aminobutyric acid, citrulline, β-alanine, α-ethylglycine, α-propylglycine and norleucine.

[0052] The terms "antibody", "immunoglobulin" or "Ig" are used interchangeably herein and in the broadest sense, and specifically encompass, for example, single monoclonal antibodies (including agonists, antagonists, neutralizing antibodies, full-length or intact monoclonal antibodies), antibody compositions with multiple or single epitope specificity, polyclonal antibodies or monovalent antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies, as long as they exhibit the desired biological activity), which are formed from at least two intact antibodies, single-chain antibodies, and antibody fragments. Antibodies can be human, humanized, chimeric and / or affinity mature, as well as antibodies from other species such as mice and rabbits.

[0053] The term "antibody" is intended to include polypeptide products of B cells within the class of immunoglobulin polypeptides, which are capable of binding to a specific antigen and are composed of two pairs of identical polypeptide chains, wherein each pair of polypeptide chains has one heavy chain (about 50-70 kDa) and one light chain (about 25 kDa), each amino-terminal portion of each chain includes a variable region of about 100 to about 130 or more amino acids, and each carboxyl-terminal portion of each chain includes a constant region. See Borrebaeck (ed.) (1995) Antibody Engineering, 2nd ed., Oxford University Press.; Kuby (1997) Immunology, 3rd ed., WH Freeman and Company, New York. Antibodies also include, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinant antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, camelized antibodies, chimeric antibodies, intrabodies, anti-idiotypic (anti-Id) antibodies, and functional fragments thereof, wherein the functional fragment refers to a portion of an antibody heavy chain or light chain polypeptide that retains some or all of the binding activity of the antibody from which the fragment is derived. Non-limiting examples of functional fragments of antibodies include single-chain Fv (scFv) (e.g., including monospecific or bispecific), Fab fragments, F(ab') fragments, F(ab)2 fragments, F(ab')2 fragments, disulfide-linked Fv (sdFv), Fd fragments, Fv fragments, scRv-Fc, nanobodies, double antibodies, three antibodies, four antibodies and mini antibodies. In some embodiments, the antibody comprises an Fc variant with reduced or eliminated effector function. Specifically, the antibodies provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, such as antigen binding domains or molecules containing antigen binding sites that bind to antigens (e.g., one or more complementary determining regions (CDRs) of anti-CD56 antibodies or anti-SIRPα antibodies).Such antibody fragments are described, for example, in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1989); Myers (ed.), Molec. Biology and Biotechnology: A Comprehensive Desk Reference, New York: VCH Publisher, Inc.; Huston et al., Cell Biophysics 1993, 22, 189-224; Plückthun and Skerra, Meth. Enzymol. 1989, 178, 497-515; and Day, Advanced Immunochemistry, 2nd edition, Wiley-Liss, Inc., New York, NY (1990). The antibodies provided herein can be any type of immunoglobulin molecule (e.g., IgG, IgE, IgM, IgD, IgA and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or any subclass (e.g., IgG2a and IgG2b).

[0054] The term "antigen" refers to a predetermined target to which an antibody can selectively bind. The target antigen can be a polypeptide, carbohydrate, nucleic acid, lipid, hapten or fragment thereof, or other naturally occurring or synthetic compound. In one embodiment, the target antigen is a polypeptide.

[0055] The terms "antigen-binding fragment," "antigen-binding domain," and "antigen-binding region" refer to a portion of an antibody comprising the amino acid residues (e.g., the complementarity determining region (CDR)) that interacts with an antigen (e.g., a polypeptide, carbohydrate, nucleic acid, lipid, hapten or fragment thereof, or other naturally occurring or synthetic compound) and confers specificity and affinity to the binding agent for the antigen.

[0056] The terms "specific binding", "specifically binds to", or "has specificity for a particular polypeptide or an epitope on a particular polypeptide target" may be defined as, for example, a polypeptide having at least about 10 - 4 M, at least about 10 -5 M, at least about 10 -6 M, at least about 10 -7 M, at least about 10 -8 M, at least about 10 -9 M, at least about 10 -10M, at least about 10 -11 M or at least about 10 -12 The dissociation constant (K d In one embodiment, the term "specific binding" refers to the binding of a molecule to a specific polypeptide or an epitope on a specific polypeptide without binding to any other polypeptide or polypeptide epitope.

[0057] The 4-chain antibody unit is a heterotetrameric glycoprotein consisting of two identical light chains (L) and two identical heavy chains (H). In the case of IgG, the 4-chain unit is usually about 150,000 daltons. Each L chain is connected to the H chain by a covalent disulfide bond, and the two H chains are connected to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has a variable domain (VH) at the N-terminus, followed by three constant domains (CH) of the α and γ chains and four CH domains of the μ and ε isotypes. The N-terminus of each L chain has a variable domain (VL), followed by a constant domain (CL) at the other end. VL is aligned with VH, and CL is aligned with the first constant domain (CH1) of the heavy chain. Specific amino acid residues are considered to form the interface between the light chain variable domain and the heavy chain variable domain. VH and VL pair together to form a single antigen binding site. For the structure and properties of different classes of antibodies, see, e.g., Basic and Clinical Immunology, 8th ed., Stites et al. (eds.), Appleton & Lange, Norwalk, CT, 1994, p. 71 and Chapter 6.

[0058] The term "variable region" or "variable domain" refers to a part of the light chain or heavy chain of an antibody, which is usually located at the amino terminal of the light chain or heavy chain, and the heavy chain length is about 120 to 130 amino acids and the light chain length is about 100 to 110 amino acids, and is used for the binding and specificity of each specific antibody to its specific antigen. The variable region of the heavy chain may be referred to as "VH". The variable region of the light chain may be referred to as "VL". The term "variable" refers to the fact that certain segments of the variable region differ greatly in sequence between antibodies. The V region mediates antigen binding and defines the specificity of a specific antibody to its specific antigen. However, variability is not evenly distributed over the 110 amino acid span of the variable region. Instead, the V region is composed of a less variable (e.g., relatively unchanged) segment called a framework region (FR) of about 15-30 amino acids, which is separated by a shorter region of greater variability (e.g., extreme variability) called a "hypervariable region", each of which is about 9-12 amino acids long. The variable regions of heavy and light chains each contain four FRs, mainly in a β-folded configuration, connected by three hypervariable regions, which form a loop connecting the β-folded structure, and in some cases form a part of the β-folded structure. The hypervariable regions in each chain are closely held together by FRs, and together with the hypervariable regions from another chain, contribute to the formation of the antigen binding site of the antibody (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition Public Health Service, National Institutes of Health, Bethesda, MD, 1991). The constant region does not directly participate in the binding of the antibody to the antigen, but exhibits various effector functions, such as antibody participation in antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The variable region sequences between different antibodies vary greatly. The variability of the sequence is concentrated in the CDR, and the less variable part in the variable region is called the framework region (FR). The CDRs of light and heavy chains are primarily responsible for the interaction of the antibody with the antigen. In a specific embodiment, the variable region is a human variable region.

[0059] The term "variable region residue numbering as in Kabat" or "amino acid position numbering as in Kabat" and variants thereof refer to the numbering system for heavy chain variable regions or light chain variable regions used for antibody compilations in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition Public Health Service, National Institutes of Health, Bethesda, MD. (1991). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids, which correspond to shortening of the FR or CDR of the variable domain or insertion therein. For example, the heavy chain variable domain may include a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and an inserted residue after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c, etc. according to Kabat). For a given antibody, the Kabat numbering of the residues can be determined by aligning the homology region of the antibody sequence with the "standard" Kabat numbering sequence. When referring to residues in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain), the Kabat numbering system is generally used (e.g., Kabat et al. Sequences of Immunological Interest. 5th ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). When referring to residues in the constant region of an immunoglobulin heavy chain, the "EU numbering system" or "EU index" is generally used (e.g., the EU index reported in Kabat et al., supra). The "EU index as in Kabat" refers to the residue numbering of the human IgG 1EU antibody. Other numbering systems have been described, including, for example, AbM, Chothia, Contact, IMGT, and AHon.

[0060] A "complete" antibody is an antibody that comprises an antigen binding site as well as CL and at least heavy chain constant regions CH1, CH2 and CH3. The constant region may comprise a human constant region or an amino acid sequence variant thereof. Preferably, the complete antibody has one or more effector functions.

[0061] The term "antibody fragment" refers to a portion of an intact antibody, preferably the antigen binding region or variable region of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies and diabodies (see, e.g., Holliger et al., Proc. Natl. Acad. Sci. USA 1993, 90, 6444-8; Lu et al. J. Biol. Chem. 2005, 280, 19665-72; Hudson et al. Nat. Med. 2003, 9, 129-134; WO 20030105444). 93 / 11161; and U.S. Pat. Nos. 5,837,242 and 6,492,123); single-chain antibody molecules (see, e.g., U.S. Pat. Nos. 4,946,778; 5,260,203; 5,482,858; and 5,476,786); dual variable domain antibodies (see, e.g., U.S. Pat. No. 7,612,181); single variable domain antibodies (sdAbs) (see, e.g., Woolven et al., Immunogenetics 1999, 50, 98-101 Streltsov et al., Proc. Natl. Acad. Sci. USA 2004, 101, 12444-12449); and multispecific antibodies formed from antibody fragments.

[0062] A "functional fragment", "binding fragment" or "antigen-binding fragment" of an antibody refers to a molecule that exhibits at least one biological function possessed by an intact antibody, wherein the function at least includes binding to a target antigen.

[0063] When used to refer to antibodies, the term "heavy chain" refers to a polypeptide chain of about 50-70 kDa, wherein the amino terminal portion comprises a variable region of about 120-130 or more amino acids, and the carboxyl terminal portion comprises a constant region. Based on the amino acid sequence of the heavy chain constant region, the constant region can be one of five different types (e.g., isotypes), referred to as alpha (α), delta (δ), epsilon (ε), gamma (γ) and mu (μ). The sizes of different heavy chains are different: α, δ and γ contain about 450 amino acids, while μ and ε contain about 550 amino acids. When combined with a light chain, these different types of heavy chains produce five well-known antibody classes (e.g., isotypes), respectively IgA, IgD, IgE, IgG and IgM, including four subclasses of IgG, i.e. IgG1, IgG2, IgG3 and IgG4. The heavy chain can be a human heavy chain.

[0064] When used to refer to antibodies, the term "light chain" refers to a polypeptide chain of about 25 kDa, wherein the amino terminal portion comprises a variable region of about 100 to about 110 or more amino acids, and the carboxyl terminal portion comprises a constant region. The approximate length of the light chain is 211 to 217 amino acids. Based on the amino acid sequence of the constant domain, there are two different types, called kappa (κ) or lambda (λ). Light chain amino acid sequences are well known in the art. The light chain can be a human light chain.

[0065] The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, e.g., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in small amounts, and each monoclonal antibody generally recognizes a single epitope on an antigen. In specific embodiments, a "monoclonal antibody" as used herein is an antibody produced by a single hybridoma or other cell, wherein the antibody binds only to an epitope of βklotho, as determined by, e.g., ELISA or other antigen binding or competitive binding assays known in the art. The term "monoclonal" is not limited to any particular method for preparing an antibody. For example, the monoclonal antibodies useful in the present disclosure may be prepared by the hybridoma method first described by Kohler et al., Nature 1975, 256, 495, or may be prepared using recombinant DNA methods in bacteria, eukaryotic animals or plant cells (see, e.g., U.S. Pat. No. 4,816,567). "Monoclonal antibodies" can also be isolated from phage antibody libraries using techniques such as those described in Clackson et al., Nature 1991, 352, 624-628 and Marks et al., J. Mol. Biol. 1991, 222, 581-597. Other methods for preparing clonal cell lines and monoclonal antibodies expressed therefrom are well known in the art (see, e.g., Short Protocols in Molecular Biology, (2002) 5th edition, Ausubel et al., ed., John Wiley and Sons, New York, Chapter 11). Exemplary methods for producing monoclonal antibodies are provided in the Examples herein.

[0066] The "humanized" form of non-human (e.g., mouse) antibodies is a chimeric antibody comprising a human immunoglobulin (e.g., a receptor antibody), wherein the native CDR residues are replaced by corresponding CDR residues with desired specificity, affinity and ability from non-human species (e.g., donor antibodies), such as mice, rats, rabbits or non-human primates. In some cases, one or more FR region residues of a human immunoglobulin are replaced by corresponding non-human residues. In addition, a humanized antibody may include residues that are not present in a receptor antibody or in a donor antibody. These modifications are made to further improve antibody performance. A humanized antibody heavy chain or light chain may include substantially all variable regions in at least one or more variable regions, wherein all or substantially all CDRs correspond to the CDRs of non-human immunoglobulins, and all or substantially all FRs are those of human immunoglobulin sequences. In certain embodiments, a humanized antibody will include at least a portion of an immunoglobulin constant region (Fc), typically a constant region of a human immunoglobulin. For further details, see Jones et al., Nature 1986, 321, 522-525; Riechmann et al., Nature 1988, 332, 323-329; Presta, Curr. Opin. Biotechnol. 1992, 3, 394-398; Carter et al., Proc. Natl. Acad. Sci. USA 1992, 89, 4285-4289; and U.S. Pat. Nos. 6,800,738, 6,719,971, 6,639,055, 6,407,213 and 6,054,297.

[0067] "Human antibody" is an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human and / or prepared using any technique disclosed herein for preparing human antibodies. This definition of human antibody explicitly excludes humanized antibodies comprising non-human antigen binding residues. Human antibodies can be produced using various techniques known in the art, including phage display libraries (Hoogenboom and Winter, J.Mol.Biol.1991, 227, 381; Marks et al., J.Mol.Biol.1991, 222, 581) and yeast display libraries (Chao et al., Nature Protocols 2006, 1, 755-768). Also useful for preparing human monoclonal antibodies are Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, 77 (1985); Boerner et al., J.Immunol.1991, 147, methods described in 86-95. See also van Dijk and van de Winkel, Curr. Opin. Pharmacol. 2001, 5, 368-374. Human antibodies can be prepared by administering an antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled, such as a mouse (see, e.g., Jakobovits, Curr. Opin. Biotechnol. 1995, 6, 561-566; Brüggemann and Taussing, Curr. Opin. Biotechnol. 1997, 8, 455-458; and about the XENOMOUSE TM Nos. 6,075,181 and 6,150,584 of the US Patent Application Publication No. 6,075,181 (US Pat. No. 6,150,584) for human antibodies generated by human B cell hybridoma technology. See also, e.g., Li et al. Proc. Natl. Acad. Sci. USA 2006, 103, 3557-3562.

[0068] "CDR" refers to one of the three hypervariable regions (H1, H2 or H3) within the non-framework region of the VH β-fold framework of an immunoglobulin (Ig or antibody), or one of the three hypervariable regions (L1, L2 or L3) within the non-framework region of the VL β-fold framework of an antibody. Thus, CDRs are variable region sequences interspersed within the framework region sequences. CDR regions are well known to those skilled in the art and have been defined by, for example, Kabat as hypervariable regions within antibody variable (V) domains. Kabat et al., J. Biol. Chem. 1977, 252, 6609-6616; Kabat, Adv. Protein Chem. 1978, 32, 1-75. CDR region sequences are also structurally defined by Chothia as those residues that are not part of the conserved β-fold framework and are therefore able to adapt to different conformations. Chothia and Lesk, J. Mol. Biol. 1987, 196, 901-917. Both terms are recognized in the art. CDR region sequences have also been defined by AbM, Contact and IMGT. The position of CDRs within the variable region of standard antibodies has been determined by comparison of multiple structures. Al-Lazikani et al., J. Mol. Biol. 1997, 273, 927-948; Morea et al., Methods. 2000, 20, 267-279. Because the number of residues in the hypervariable region varies in different antibodies, the additional residues relative to the standard position are usually numbered with a, b, c, etc. next to the residue number in the standard variable region numbering scheme. Al-Lazikani et al., supra (1997). This nomenclature is also well known to those skilled in the art.

[0069] The term "hypervariable region", "HVR" or "HV" when used herein refers to a region in an antibody variable region that is highly variable in sequence and / or forms a structurally determined loop. Typically, an antibody comprises six hypervariable regions; three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Descriptions of many hypervariable regions are in use and are covered herein. The Kabat complementarity determining region (CDR) is based on the variability of the sequence and is the most commonly used (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Chothia refers to the position of the structural loop. See, e.g., Chothia and Lesk, J. Mol. Biol. 1987, 196, 901-917. When numbered using the Kabat numbering convention, the ends of the Chothia CDR-H1 loop vary between H32 and H34, depending on the length of the loop (this is because the Kabat numbering scheme places the insertion at H35A and H35B; if both 35A and 35B are absent, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable region represents a compromise between the Kabat CDR and Chothia structural loops and is used by Oxford Molecular's AbM antibody modeling software (see, e.g., Martin, in Antibody Engineering, Vol. 2, Chapter 3, Springer Verlag). The "contact" hypervariable region is based on an analysis of existing complex crystal structures. The residues of each of these hypervariable regions or CDRs are as follows.

[0070] The term "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain, including, for example, a native sequence Fc region, a recombinant Fc region, and a variant Fc region. Although the boundaries of an immunoglobulin heavy chain Fc region may be different, a human IgG heavy chain Fc region is generally defined as extending from position Cys226 or from an amino acid residue at position Pro230 to its carboxyl terminus. The C-terminal lysine in the Fc region (residue 447 according to the EU numbering system) can be removed, for example, during production or purification of antibodies or by recombinant engineering of nucleic acids encoding antibody heavy chains. Therefore, the composition of a complete antibody can include an antibody population in which all K447 residues are removed, an antibody population in which no K447 residue is removed, and an antibody population with an antibody mixture containing or not containing the K447 residue.

[0071] The term "DAR" refers to the drug-antibody ratio of an oligonucleotide-antibody conjugate, more specifically the immunomodulatory polynucleotide-antibody ratio. In some cases, for example, an oligonucleotide-antibody conjugate may be described herein as having a DAR of 1 or a DAR1 conjugate, wherein the oligonucleotide-antibody ratio is 1 to 1. In other cases, an oligonucleotide-antibody conjugate may be described herein as having a DAR of 2 or a DAR2 conjugate, wherein the oligonucleotide-antibody ratio is 2 to 1.

[0072] As used herein, the terms "comprising," "containing," and "including" are used in an open, non-limiting sense. It should also be understood that the aspects and embodiments of the invention described herein may include "consisting of" and / or "consisting essentially of" the aspects and embodiments.

[0073] It should be understood that, regardless of whether the term "about" is explicitly used, each quantity given herein is intended to refer to the actual given value, and is also intended to refer to the approximate value to the given value that can be reasonably inferred based on ordinary skill in the art, including equivalent values ​​and approximate values ​​resulting from experimental and / or measurement conditions to the given value.

[0074] As used herein, "carrier" includes pharmaceutically acceptable carriers, excipients or stabilizers that are nontoxic to cells or mammals exposed thereto at the dosages and concentrations used. Typically, a physiologically acceptable carrier is a pH buffered aqueous solution. Non-limiting examples of physiologically acceptable carriers include, but are not limited to, buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN TM , polyethylene glycol (PEG) and PLURONICS TM .

[0075] The term "effective amount" or "therapeutically effective amount" of a substance as used herein is at least the minimum concentration required to bring about a measurable improvement (e.g., such as toward a treatment endpoint) or prevent a particular condition. The effective amount herein can vary according to factors such as the patient's disease state, age, sex, and weight, and the ability of the substance to elicit a desired response in an individual. An effective amount is also an amount in which any toxic or harmful effects of treatment are surpassed by the beneficial effects of treatment. For cancer, an effective amount includes an amount sufficient to shrink a tumor and / or reduce tumor growth rate (e.g., inhibit tumor growth) or prevent or delay other unnecessary cell proliferation in cancer or reach a specific threshold of a response standard. In some embodiments, an effective amount is an amount sufficient to delay cancer development. In some embodiments, an effective amount is an amount sufficient to prevent or delay recurrence. In some embodiments, an effective amount is an amount sufficient to reduce the recurrence rate of an individual. An effective amount can be administered in one or more administrations. An effective amount of a drug or composition can: (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, block, slow down to some extent and preferably stop the infiltration of cancer cells into peripheral organs; (iv) inhibit (i.e., slow down to some extent and preferably stop) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay the occurrence and / or recurrence of tumors; (vii) reduce the recurrence rate of tumors, and / or (viii) alleviate one or more symptoms associated with cancer to some extent. An effective amount can be administered in one or more administrations. For the purposes of this disclosure, an effective amount of a drug, compound or pharmaceutical composition is an amount sufficient to directly or indirectly achieve a preventive or therapeutic treatment. As understood in clinical situations, an effective amount of a drug, compound or pharmaceutical composition may or may not be achieved in combination with another drug, compound or pharmaceutical composition. Therefore, an "effective amount" can be considered in the context of administering one or more therapeutic agents, and if a desired result can be achieved or achieved in combination with one or more other agents, then a single agent can be considered to be administered in an effective amount.

[0076] “Package insert” means instructions customarily included in commercial pharmaceutical packages that contain information regarding the indications, usage, dosage, administration, contraindications, other drugs combined with the packaged product, and / or warnings concerning the use of such drugs.

[0077] The terms "protein," "polypeptide," and "peptide" are used herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be interspersed with non-amino acids. These terms also encompass amino acid polymers that have been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification (such as conjugation with a labeling component). Typically, proteins used herein will have a molecular weight of at least about 5-20 kDa, or at least about 20-100 kDa, or at least about 100 kDa. Also included in the definition are, for example, one or more analogs containing amino acids (including, for example, non-natural amino acids, etc.) and other modified proteins known in the art.

[0078] "Pharmaceutically acceptable salt" refers to a salt form that is non-toxic, biologically tolerable, or biologically suitable for administration to a subject. See generally Berge et al. (1977) J. Pharm. Sci. 66, 1. Specific pharmaceutically acceptable salts are those that are pharmacologically effective and suitable for contact with the tissues of a subject without producing excessive toxicity, irritation, or allergic reactions. Pharmaceutically acceptable salts include, but are not limited to, acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc.; or acid addition salts formed with organic acids such as acetic acid, oxalic acid, propionic acid, succinic acid, maleic acid, tartaric acid, etc. These salts may be derived from inorganic or organic acids. Non-limiting examples of pharmaceutically acceptable salts include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrogenphosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, octanoates, acrylates, formates, isobutyrates, hexanoates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyrates, -1,4-dioic acid salt, hexyne-1,6-dioic acid salt, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, methylsulfonate, propylsulfonate, benzenesulfonate, xylenesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, γ-hydroxybutyrate, glycolate, tartrate and mandelate. In some embodiments, when the acidic proton present in the parent compound is replaced by a metal ion such as an alkali metal ion, an alkaline earth metal ion or an aluminum ion; or when coordinated with an organic base, a pharmaceutically acceptable salt is formed. Salts derived from pharmaceutically acceptable organic non-toxic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines (including naturally occurring substituted amines), cyclic amines, and basic ion exchange resins such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-diethylaminoethanol, trishydroxymethylaminomethane, trimethylamine, dicyclohexylamine, caffeine, procaine, hydrobromide, choline, betaine, ethylenediamine, glucosamine, N-ethylglucosamine, N-methylglucosamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, amino acids such as lysine, arginine, histidine, etc. Examples of pharmaceutically acceptable base addition salts include base addition salts derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts, and the like. In some embodiments, the organic non-toxic base is an L-amino acid, such as L-lysine and L-arginine, tromethamine, N-ethylglucosamine and N-methylglucosamine. Acceptable inorganic bases include, but are not limited to, aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, and the like.Lists of other suitable pharmaceutically acceptable salts can be found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985.

[0079] "Solvates" are formed by the interaction of a solvent and a compound. Suitable solvents include, for example, water and alcohols (eg, ethanol). Solvates include hydrates having any compound / water ratio, such as monohydrates, dihydrates, and hemihydrates.

[0080] "Subject," "patient," or "individual" refers to a human. The individual can be male or female and can be of any suitable age, including infants, juveniles, adolescents, adults, and elderly subjects.

[0081] "Cancer" or "tumor" refers to cells that have typical characteristics of oncogenic cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Cancer cells are usually present in the form of solid tumors, which can be detected based on the mass of the tumor, for example, by procedures such as CAT scans, MR imaging, X-rays, ultrasound or palpation, and / or due to the expression of one or more cancer-specific antigens in a sample that can be obtained from the patient. In some embodiments, solid tumors need not have a measurable size. Cancer cells can be in the form of liquid tumors, which can exist alone or spread throughout the animal's body. The terms "disseminated tumors" and "liquid tumors" as used herein are used interchangeably and include, but are not limited to, leukemias and lymphomas and other blood cell cancers.

[0082] The terms "cancer recurrence" and "cancer relapse" are used interchangeably and refer to the reappearance of signs, symptoms, or disease after remission. Recurrent cancer cells may reappear at the same site of the primary tumor or at another location, such as in a secondary cancer. Cancer cells may reappear in the same disease form as the primary cancer or in a different disease form. For example, in some embodiments, the primary cancer is a solid tumor and the recurrent cancer is a liquid tumor. In other embodiments, the primary cancer is a liquid tumor and the recurrent cancer is a solid tumor. In other embodiments, both the primary cancer and the recurrent cancer are solid tumors, or both are liquid tumors. In some embodiments, the recurrent tumor expresses at least one tumor-associated antigen that is also expressed by the primary tumor.

[0083] As used herein, the term "refractory cancer" refers to a cancer that does not respond to treatment, such as a cancer that is resistant at the start of treatment (e.g., treatment with immunotherapy) or a cancer that may become resistant during treatment. The terms "respond," "response," or "responsiveness" refer to an anti-cancer response, such as a reduction in tumor size or inhibition of tumor growth. These terms may also refer to an improved prognosis, for example, reflected in an increase in the time to recurrence, i.e., the time to first recurrence review for a second primary cancer as the first event or death without evidence of recurrence, or an increase in overall survival, i.e., the time from treatment to death from any cause. Responding or producing a response means reaching a beneficial endpoint when exposed to a stimulus. Alternatively, when exposed to a stimulus, negative or harmful symptoms are minimized, alleviated, or attenuated. It should be understood that assessing the likelihood that a tumor or subject will show a favorable response is equivalent to assessing the likelihood that a tumor or subject will not show a favorable response (i.e., show a lack of response or no response).

[0084] Cancer as used herein includes, but is not limited to, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, oral or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, bile duct cancer, small intestine or appendix cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, blood tissue cancer, B cell cancer (e.g., multiple myeloma), Waldenstrom's macroglobulinemia, heavy chain disease (e.g., α chain disease, γ chain disease, and μ chain disease), benign monoclonal gammopathy, and immune cell amyloidosis, etc. Other non-limiting examples of cancer types suitable for use in the methods of the invention include human sarcomas and carcinomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct cancer, liver cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms tumor, cervical cancer, bone cancer, brain cancer, tumors, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial cancers, gliomas, astrocytomas, medulloblastomas, craniopharyngiomas, ependymomas, pinealomas, hemangioblastomas, acoustic neuromas, oligodendrogliomas, meningiomas, melanomas, neuroblastomas, retinoblastomas; leukemias, such as acute lymphocytic leukemia and acute myeloid leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia); chronic leukemias (chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia); as well as polycythemia vera, lymphomas (Hodgkin's and non-Hodgkin's), multiple myeloma, Waldenstrom's macroglobulinemia, and heavy chain disease. In some embodiments, the cancer is epithelial in nature, including but not limited to bladder cancer, breast cancer, cervical cancer, colon cancer, gynecological cancer, kidney cancer, laryngeal cancer, lung cancer, oral cancer, head and neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, or skin cancer. In other embodiments, the cancer is breast cancer, prostate cancer, lung cancer, or colon cancer. In other embodiments, the epithelial cancer is non-small cell lung cancer, non-papillary renal cell carcinoma, cervical cancer, ovarian cancer (e.g., high-grade serous ovarian cancer), or breast cancer. Epithelial cancer can be characterized by a variety of other ways, including but not limited to serous, endometrioid, mucinous, clear cell, Brenner cell, or undifferentiated.

[0085] The term "cancer therapy" or "cancer therapeutic agent" as used herein refers to a therapy or agent that can exert an anti-tumor effect or has anti-tumor activity. This anti-tumor effect or anti-tumor activity can be manifested as a decrease in tumor cell proliferation rate, vitality or metastatic activity. One possible way to demonstrate anti-tumor activity is to show a decrease in the growth rate of abnormal cells that appear during treatment or a stable or reduced tumor size. This activity can be evaluated using an acceptable in vitro or in vivo tumor model, including but not limited to a xenograft model, an allograft model, an MMTV model, and other models known in the art for studying anti-tumor activity.

[0086] The term "treat / treating / treatment" is intended to include alleviating or eliminating an illness, condition, or disease, or one or more symptoms associated with an illness, condition, or disease; or alleviating or eliminating the cause of the illness, condition, or disease itself. In some embodiments, cancer treatment results in a complete response (CR) or a partial response (PR). In some embodiments, cancer treatment results in a CR, PR, or stable disease (SD). Exemplary criteria for CR, PR, and SD are provided herein.

[0087] The terms "prevent", "preventing" and "prevention" are intended to include methods of delaying and / or preventing the onset of a disorder, condition or disease and / or its attendant symptoms; methods of preventing a subject from developing a disorder, condition or disease; or methods of reducing the risk of a subject developing a disorder, condition or disease.

[0088] By "optional" or "optionally" is meant that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not occur. For example, "optionally substituted alkyl" encompasses both "alkyl" and "substituted alkyl" as defined herein. It will be understood by those skilled in the art that, with respect to any group containing one or more substituents, such groups are not intended to introduce any substitution or substitution pattern that is sterically impractical, synthetically unfeasible, and / or inherently unstable. It should also be understood that, where a group or moiety is optionally substituted, the present disclosure includes embodiments in which the group or moiety is substituted and embodiments in which the group or moiety is not substituted.

[0089] The term "Q tag" as used herein refers to a portion of a polypeptide comprising a glutamine residue, which, after a transglutaminase-mediated reaction with a compound containing -NH2 amine, provides a conjugate comprising the polypeptide portion, wherein the glutamine residue includes a side chain modified to include an amide bonded to the compound. Q tags are known in the art. Non-limiting examples of Q tags are LLQGG (SEQ ID NO: 172), GGGLLQGG (SEQ ID NO: 173), RPQGF (SEQ ID NO: 47) and RPQGFGPP (SEQ ID NO: 49). In some embodiments, the Q tag is attached to the C-terminus of the heavy chain of an antibody. In some embodiments, the Q tag is attached to the light chain of an antibody. In some embodiments, the Q tag is naturally occurring. For example, a mutation of N297 to N297A exposes Q295 of the antibody, where conjugation may occur (numbering according to the EU index, e.g., as listed in Edelman, GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969) and Kabat, EA et al., Sequences of proteins of immunological interest. 5th ed. - US Department of Health and Human Services, NIH publication n°91-3242, pp 662, 680, 689 (1991)). In some embodiments, the Q tag is within the Fc domain of the antibody.

[0090] II. Methods of treating cancer

[0091] Provided herein is a method for treating cancer in an individual, comprising administering a conjugate comprising an anti-CD22 antibody and an immunomodulatory oligonucleotide (e.g., a CpG oligonucleotide). In some embodiments, cancer is a tumor, such as an advanced or metastatic solid tumor. In some embodiments, the tumor is a solid tumor that is histologically or cytologically confirmed to be inoperable, locally advanced, metastatic, or recurrent. In some embodiments, the conjugate is administered to an individual with a solid tumor and the individual's cancer has progressed or the individual is intolerant to standard therapy or the cancer has no standard therapy available. In some embodiments, the cancer to be treated is skin melanoma, breast cancer, ovarian cancer, colorectal cancer, non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC), head and neck squamous cell carcinoma (HNSCC), Merkel cell carcinoma, hepatocellular carcinoma (HCC), cervical squamous cell carcinoma, urothelial carcinoma, endometrial carcinoma, gastroesophageal adenocarcinoma, or bile duct cancer. In some embodiments, the cancer to be treated is triple-negative breast cancer, melanoma, ovarian cancer, or colon cancer. In some embodiments, the presence or level of PD-L1 in the tumor tissue of the cancer to be treated is tested. In some embodiments, the conjugate is administered to an individual at a dose between 0.1 mg / kg and 60 mg / kg (e.g., 0.1, 0.3, 1.0, 3.0, 10.0, 30.0, or 60.0 mg / kg). In some embodiments, the conjugate is administered to an individual at a dose between 0.1-1 mg / kg, between 1-10 mg / kg, between 10-30 mg / kg, or between 30-60 mg / kg. In some embodiments, the dose administered is 0.1 mg / kg. In some embodiments, the dose administered is 0.3 mg / kg. In some embodiments, the dose administered is 1.0 mg / kg. In some embodiments, the dose administered is 3.0 mg / kg. In some embodiments, the dose administered is 10.0 mg / kg. In some embodiments, the dose administered is 30.0 mg / kg. In some embodiments, the dose administered is 60.0 mg / kg. In some embodiments, the dose is administered Q1W (weekly) or about once every 7 days. In some embodiments, the dose is administered Q2W (every two weeks) or about once every 14 days. In some embodiments, the dose is administered Q3W (every 3 weeks) or about once every 21 days. In some embodiments, the dose is administered Q4W (once every 4 weeks), about once every 28 days, or about once a month. In some embodiments, the dose is administered once, twice, 3 times, 4 times, 5 times, or more than 5 times during the course of treatment. In some embodiments, the individual is a human. In some embodiments, the conjugate is administered intravenously (IV) to the individual.In some embodiments, the conjugate is administered as a formulation comprising the conjugate at a concentration of 30 mg / mL, 20 mM citrate, 150 mM L-arginine, 50 mM NaCl, and 0.02% polysorbate 80 (w / v); wherein the pH of the formulation is 6.5.

[0092] In some embodiments, treatment according to the methods of the present disclosure results in a complete response (CR) in an individual. In some embodiments, CR refers to the disappearance of all non-lymph node target lesions and the reduction of the short axis of any pathological lymph node designated as a target lesion to <10 mm.

[0093] In some embodiments, treatment according to the methods of the present disclosure results in a partial response (PR) in an individual. In some embodiments, a PR is at least a 30% reduction in the sum of diameters of all target lesions relative to the baseline sum of diameters.

[0094] In some embodiments, treatment according to the methods of the present disclosure results in stable disease (SD) in an individual. In some embodiments, SD refers to a reduction in target lesions that is not sufficient to qualify for PR or CR (e.g., as described above) and an increase in target lesions that is not sufficient to qualify for progressive disease (PD). In some embodiments, PD refers to a minimum sum of all target lesion diameters recorded at or after baseline as a reference, an increase of at least 20% in the sum of all measured target lesion diameters, and a sum showing an absolute increase of at least 5 mm.

[0095] In some embodiments, the methods of the present disclosure are used to treat inoperable, locally advanced, metastatic, and / or recurrent cancers (eg, solid tumors).

[0096] In some embodiments, locally advanced or late stage cancer refers to cancer that has spread only to nearby tissues or lymph nodes. For example, with locally advanced breast cancer, the cancer may involve the skin of the breast or the muscles underneath the chest, or involve multiple regional lymph nodes (lymph nodes located in the soft tissue above and below the armpit or collarbone). A second example is lung cancer; locally advanced lung cancer means that the cancer has spread to the airways, chest wall, or membrane surrounding the lungs (pleura).

[0097] In some embodiments, metastatic cancer refers to cancer that has spread to distant parts of the body. For example, the most common sites of metastatic breast cancer spread are bones, lungs, brain, and liver. A second example is metastatic lung cancer, where the cancer has spread to the brain, bones, liver, adrenal glands, other parts of the lung, or another lung.

[0098] In some embodiments, the cancer (eg, solid tumor) treated by the methods of the present disclosure is documented histologically and / or cytologically.

[0099] In some embodiments, in the immunomodulatory oligonucleotide-antibody conjugates herein, the oligonucleotide and the antibody are linked together by a linking moiety. In some embodiments, an antibody can be conjugated to an oligonucleotide (DAR 1), such as Figure 1 In some embodiments, the oligonucleotide-antibody conjugate is a conjugate comprising an antibody or an antigen-binding fragment thereof and one or more immunomodulatory oligonucleotides (P), wherein the antibody or antigen-binding fragment is linked to one or more Q-tag peptides (Q) comprising at least one glutamine residue, wherein each immunomodulatory oligonucleotide is linked to the Q-tag peptide via an amide bond to the glutamine residue of the Q-tag peptide and a linker (L), as shown in formula (A):

[0100]

[0101] or a stereoisomer, a mixture of two or more diastereomers, a tautomer, or a mixture of two or more tautomers thereof; or a pharmaceutically acceptable salt, solvate or hydrate thereof;

[0102] in:

[0103] represents the point of attachment of each Q to the antibody or antigen-binding fragment thereof (Ab);

[0104] Each Q is independently a Q-tag peptide sequence comprising at least one glutamine residue;

[0105] Each L is independently a bond or linker moiety connected to Q through an amide bond to a glutamine residue; and

[0106] Each P is independently an immunomodulatory oligonucleotide or a pharmaceutically acceptable salt thereof.

[0107] In another aspect, the oligonucleotide is conjugated to the polypeptide via a linker. The length, rigidity, and chemical composition of the linker affect the rate of the conjugation reaction and the stability of the resulting conjugate.

[0108] Connector part (L)

[0109] In some embodiments, the linking moiety comprises polyethylene glycol (PEG). In some embodiments, PEG comprises about 10-50 ethylene glycol units. In some embodiments, the linking moiety is an aliphatic chain.

[0110] For Formula (A), the linking moiety is represented by L. In some embodiments, the linker L comprises an oligoethylene glycol or polyethylene glycol moiety. In certain embodiments, the linker L is a poly(ethylene glycol) having the structure of groups, among which represents the point of attachment to oligonucleotide P, and The attachment point to the glutamine residue of the Q tag is indicated.

[0111] In other embodiments, linker L has the structure of groups, among which represents the point of attachment to oligonucleotide P, and In some embodiments, L 1 In some embodiments, L 1 is unsubstituted alkyl. In some embodiments, L 1 is independently unsubstituted C 1-6 In some embodiments, each L 1 In some embodiments, L 1 is independently substituted alkyl. 1 Independently substituted C 1-6 In some embodiments, L 1 is C substituted with one or more substituents selected from the group consisting of 1-6 Alkyl: alkoxy, acyl, acyloxy, alkoxycarbonyl, carbonylalkoxy, acylamino, amino, aminoacyl, aminocarbonylamino, aminocarbonyloxy, cycloalkyl, cycloalkenyl, cyano, azido, halo, hydroxy, nitro, carboxyl, thiol, sulfanyl, alkyl, alkenyl, alkynyl, heterocyclyl, aminosulfonyl, sulfonylamino, sulfonyl and oxo.

[0112] In some embodiments, L 2 In some embodiments, L 2 is an unsubstituted or substituted alkyl group.

[0113] In some embodiments, L 3 In some embodiments, L 3 is a linker moiety. In some embodiments, the linker moiety is an unsubstituted or substituted alkyl. In some embodiments, the linker moiety is independently an unsubstituted C 1-6 In some embodiments, the linker moiety is methyl or ethyl. In some embodiments, the linker moiety is independently substituted alkyl. In some embodiments, the linker moiety is independently substituted C 1-6 In some embodiments, the linker moiety is a C substituted with one or more substituents selected from the group consisting of 1-6Alkyl: alkoxy, acyl, acyloxy, alkoxycarbonyl, carbonylalkoxy, acylamino, amino, aminoacyl, aminocarbonylamino, aminocarbonyloxy, cycloalkyl, cycloalkenyl, cyano, azido, halo, hydroxy, nitro, carboxyl, thiol, sulfanyl, alkyl, alkenyl, alkynyl, heterocyclyl, aminosulfonyl, sulfonylamino, sulfonyl, and oxo. In some embodiments, the linker moiety is an amino acid residue. In some embodiments, the amino acid is selected from the group consisting of glycine, alanine, glutamic acid, and proline. In some embodiments, the linker is methyl. In some embodiments, the linker moiety is -R 5 C(O)R 6 NHR 7 -, where R 5 and R 7 is independently absent or unsubstituted or substituted alkyl, and R 6 is an amino acid residue. In some embodiments, the amino acid is selected from the group consisting of glycine, alanine, glutamic acid and proline. In some embodiments, the linker moiety is -R 3 C(O)NHR 4 -, where R 3 and R 4 is independently absent or unsubstituted or substituted alkyl. 3 is a methylene group and R 4 is -(CH2)4-. In some embodiments, R 3 is a methylene group and R 4 When there is more than one oligonucleotide sequence (i.e. p=2), the two L 1 Can be different or the same, two L 2 Can be different or the same, two L 3 Can be different or the same.

[0114] In some embodiments, m is about 3-10, about 10-15, about 15-20, about 20-25, about 25-30, about 5-16, about 15-30, about 15-25, or about 20-30. In some embodiments, m is 20, 21, 22, 23, 24, or 25.

[0115] Immunomodulatory oligonucleotides

[0116] In some embodiments, the immunomodulatory oligonucleotide comprises the structure

[0117]

[0118] or a pharmaceutically acceptable salt thereof,

[0119] in

[0120] and indicates the attachment point within the oligonucleotide;

[0121] Each T 1 independently O or S;

[0122] Each T 2 Yes - or S - ;

[0123] T 3 It is a group in represents the connection point with L, and where # indicates the point of attachment to the rest of the oligonucleotide;

[0124] Z is O or S;

[0125] U 5 ' is –H or halogen;

[0126] R 5 ' is -H or methoxy;

[0127] R c1 is -H or methoxy;

[0128] R g1 , R g2 , R g3 and R g4 is H or oxo, wherein

[0129] R 3 ' is methoxy;

[0130] R 1 C 1-4 - alkylene-hydroxyl group;

[0131] R 2 is -H or methyl; and

[0132] n is an integer from 0 to 2.

[0133] In other embodiments, the immunomodulatory oligonucleotide comprises the structure

[0134]

[0135] or a pharmaceutically acceptable salt thereof,

[0136] in

[0137] and indicates the attachment point within the oligonucleotide;

[0138] Each T 1independently O or S;

[0139] Each T 2 Yes - or S - ;

[0140] T 3 It is a group in represents the connection point with L, and where # indicates the point of attachment to the rest of the oligonucleotide;

[0141] Z is O or S;

[0142] R 5' is -H or methoxy;

[0143] R c1 is -H or methoxy;

[0144] R g1 , R g2 , R g3 and R g4 is H or oxo, wherein

[0145] R 3' It is methoxy;

[0146] R 1 C 1-4 - alkylene-hydroxyl group;

[0147] R 2 is -H or methyl; and

[0148] n is an integer from 0 to 2.

[0149] In yet other embodiments, the immunomodulatory oligonucleotide comprises the structure

[0150]

[0151] or a pharmaceutically acceptable salt thereof,

[0152] in

[0153] and indicates the attachment point within the oligonucleotide;

[0154] Each T 1 independently O or S;

[0155] Each T 2 Yes - or S - ;

[0156] T 3 It is a group in represents the connection point with L, and where # indicates the point of attachment to the rest of the oligonucleotide;

[0157] Z is O or S;

[0158] R 5 ' is -H or methoxy;

[0159] R c1 is -H or methoxy;

[0160] R g1 , R g2 , R g3 and R g4 is H or oxo, wherein

[0161] R 3 ' is methoxy;

[0162] R 1 C 1-4 - alkylene-hydroxyl group;

[0163] R 2 is -H or methyl; and

[0164] n is an integer from 0 to 2.

[0165] In some embodiments, the immunomodulatory oligonucleotide comprises an oligonucleotide sequence as shown in Table A or a pharmaceutically acceptable salt thereof. In some embodiments, the immunomodulatory oligonucleotide is an oligonucleotide as shown in Table A or a pharmaceutically acceptable salt thereof. In some embodiments, the immunomodulatory oligonucleotide comprises an oligonucleotide as shown in Table A or a pharmaceutically acceptable salt thereof. In some embodiments, the immunomodulatory oligonucleotide (P) plus a linker comprises an oligonucleotide selected from the group consisting of SEQ ID NO: 34-35 or a pharmaceutically acceptable salt thereof. In some embodiments, the immunomodulatory oligonucleotide (P) plus a linker comprises an oligonucleotide of SEQ ID NO: 34 or a pharmaceutically acceptable salt thereof. In some embodiments, the immunomodulatory oligonucleotide (P) plus a linker comprises an oligonucleotide of SEQ ID NO: 35 or a pharmaceutically acceptable salt thereof. In some embodiments, the immunomodulatory oligonucleotide (P) is an oligonucleotide selected from the group consisting of SEQ ID NO: 14-25, 141-152 and 162 or a pharmaceutically acceptable salt thereof. In some embodiments, the immunomodulatory oligonucleotide (P) comprises an oligonucleotide selected from the group consisting of SEQ ID NO: 14-25, 141-152 and 162, or a pharmaceutically acceptable salt thereof. In some embodiments, the immunomodulatory oligonucleotide (P) comprises an oligonucleotide selected from the group consisting of SEQ ID NO: 14-25, 141-152 and 162, or a pharmaceutically acceptable salt thereof.

[0166] Table A. Modified Oligonucleotides

[0167]

[0168]

[0169]

[0170] * u： 5-Bromo-2'-deoxyuridine

[0171] g： 8-Oxo-7,8-dihydro-2'-deoxyguanosine

[0172] u:5-Bromo-2'-OMe uridine

[0173] c:2'-OMe-Cytidine

[0174] t:2'-OMe-thymidine

[0175] u :2'-OMe-Uridine

[0176] u:2'-deoxyuridine

[0177] T:2'-OMOE thymidine

[0178] ts: phosphotriester linker after thymidine-PEG 24 -NH2

[0179]

[0180] t s: a phosphotriester linker after thymidine;

[0181] Lowercase: 2'-deoxynucleotide

[0182] s: phosphorothioate bond

[0183] s2: phosphorodithioate bond

[0184] c3:

[0185] s2-c3:

[0186] In some embodiments, the immunomodulatory oligonucleotide (e.g., CpG oligonucleotide) comprises an oligonucleotide sequence as shown in Table A (compound number ends with "a") or a pharmaceutically acceptable salt thereof. In some embodiments, the immunomodulatory oligonucleotide (e.g., CpG oligonucleotide) is an oligonucleotide selected from the group consisting of the oligonucleotides of Table 9 or a pharmaceutically acceptable salt thereof. In some embodiments, the immunomodulatory oligonucleotide (e.g., CpG oligonucleotide) comprises an oligonucleotide selected from the group consisting of the oligonucleotides of Table 9 or a pharmaceutically acceptable salt thereof. In some embodiments, the immunomodulatory oligonucleotide (e.g., CpG oligonucleotide) is compound 7.6a or compound 7.7a, or a pharmaceutically acceptable salt thereof. In some embodiments, the immunomodulatory oligonucleotide (e.g., CpG oligonucleotide) is compound 7.7a ​​or a pharmaceutically acceptable salt thereof.

[0187] Table 9. Modified oligonucleotide structures (containing -PEG2NH2)

[0188]

[0189] and represents the attachment point within the oligonucleotide, where Indicates the connection point with the linker L.

[0190] In some embodiments, the oligonucleotide is functionalized with a chemical tag to connect to a connecting portion. In some embodiments, the chemical tag is attached to the internucleoside bond of the oligonucleotide. In some embodiments, the chemical tag is attached to the 5' internucleoside bond. In some embodiments, the chemical tag is attached to the 3' internucleoside bond. In some embodiments, the internucleoside bond is a phosphorothioate bond. In some embodiments, the internucleoside bond is a phosphorodithioate bond. In some embodiments, the chemical tag is closer to the 5' end of the oligonucleotide than to the 3' end. In some embodiments, the chemical tag is attached to a core base.

[0191] In some embodiments of Formula (A), the linker L is of structure A group wherein m is 24, and wherein represents the point of attachment to oligonucleotide P, and represents the point of attachment to the glutamine residue of the Q tag; and wherein the oligonucleotide P comprises the following structure:

[0192]

[0193] in

[0194] and indicates the attachment point within the oligonucleotide;

[0195] Each T 1 independently O or S;

[0196] Each T 2 Yes - or S - ;

[0197] T 3 It is a group in represents the connection point with L, and where # indicates the point of attachment to the rest of the oligonucleotide;

[0198] Z is O or S;

[0199] U 5 ' is –H or halogen;

[0200] R 5 ' is -H or methoxy;

[0201] R c1 is -H or methoxy;

[0202] R g1 , R g2 , R g3 and R g4 is H or oxo, wherein

[0203] R 3 ' is methoxy;

[0204] R 1 C 1-4 - alkylene-hydroxyl group;

[0205] R 2 is -H or methyl; and

[0206] n is an integer from 0 to 2.

[0207] In some embodiments, the linker-oligonucleotide LP comprises an oligonucleotide sequence as shown in Table A or a pharmaceutically acceptable salt thereof. In some embodiments, the linker-oligonucleotide LP is a compound as shown in Table 10 or a pharmaceutically acceptable salt thereof. In some embodiments, the linker-oligonucleotide LP is compound 7.6b or compound 7.7b or a pharmaceutically acceptable salt thereof. In some embodiments, the linker-oligonucleotide LP is compound 7.6b or a pharmaceutically acceptable salt thereof. In some embodiments, the linker-oligonucleotide LP is compound 7.7b or a pharmaceutically acceptable salt thereof. In some embodiments, the linker-oligonucleotide LP comprises an oligonucleotide of SEQ ID NO: 34 or a pharmaceutically acceptable salt thereof. In some embodiments, the linker-oligonucleotide LP comprises an oligonucleotide of SEQ ID NO: 35 or a pharmaceutically acceptable salt thereof. In some embodiments, the linker-oligonucleotide LP comprises an oligonucleotide selected from the group consisting of SEQ ID NO: 3-13, 26-38, 130-140, 153-161 and 163-166 or a pharmaceutically acceptable salt thereof.

[0208] Table 10. Modified oligonucleotide structures (with -PEG2NHCOPEG 24 NH2)

[0209]

[0210] and represents the attachment point within the oligonucleotide, where Indicates the connection point with the linker L.

[0211] Anti-CD22 antibody with Q tag

[0212] In some embodiments, the oligonucleotides of the present disclosure are conjugated to anti-CD22 antibodies. In some embodiments, the oligonucleotides are conjugated to the antibodies through one or more Q tags. In some embodiments, the Q tag comprises a glutamine residue connected to the remainder of the conjugate. In other embodiments of this aspect that can be combined with any of the preceding embodiments, each Q tag independently comprises or is a peptide sequence selected from the group consisting of SEQ ID NO: 39-55. In some embodiments, each Q tag independently comprises or is a peptide sequence selected from the group consisting of the peptide sequences of Table 3. In other embodiments of this aspect, each Q tag independently comprises or is a peptide sequence selected from the group consisting of SEQ ID NO: 40-55. In yet other embodiments, each Q tag independently comprises or is a peptide sequence selected from the group consisting of SEQ ID NO: 47-49. In some embodiments, the Q tag includes LLQGG (SEQ ID NO: 172), GGGLLQGG (SEQ ID NO: 173), RPQGF (SEQ ID NO: 47) or RPQGFGPP (SEQ ID NO: 49). In some embodiments, the Q tag comprises the peptide sequence RPQGF (SEQ ID NO: 47). In certain embodiments, the Q tag comprising the peptide sequence RPQGF (SEQ ID NO: 47) is selected from the group consisting of RPQGF (SEQ ID NO: 47), RPQGFPP (SEQ ID NO: 48) and RPQGFGPP (SEQ ID NO: 49). In some embodiments, the Q tag comprises the peptide sequence RPQGFGPP (SEQ ID NO: 49).

[0213] In some embodiments, the Q tag comprises one or more sequences shown in Table 3.

[0214] Table 3. Q-tag peptide sequences

[0215]

[0216]

[0217] Certain aspects of the present disclosure relate to CD22 and anti-CD22 antibodies. In some embodiments, CD22 refers to human CD22, and the antibody binds to human CD22. CD22 is also known as Siglec-2, and CD22 genes and polypeptide sequences (e.g., human genes and polypeptide sequences) are known in the art. For example, see NCBI Gene ID No. 933 and NCBI Ref. Sequence Accession No. NP_001172028. Any anti-CD22 antibody provided herein can be used in the conjugates and methods of the present disclosure.

[0218] In some embodiments, the antibodies or conjugates of the present disclosure comprise a VH domain comprising 1, 2 or 3 CDRs of a single antibody shown in Table 4. In some embodiments, the antibodies or conjugates of the present disclosure comprise a VH domain comprising 3 CDRs of a single antibody shown in Table 4. In some embodiments, the antibodies or conjugates of the present disclosure comprise a VL domain comprising 1, 2 or 3 CDRs of a single antibody shown in Table 4. In some embodiments, the antibodies or conjugates of the present disclosure comprise a VL domain comprising 3 CDRs of a single antibody shown in Table 4. In some embodiments, the antibodies or conjugates of the present disclosure comprise a VH domain comprising 1, 2 or 3 CDRs of a single antibody shown in Table 4, and a VL domain comprising 1, 2 or 3 CDRs of a single antibody shown in Table 4. In some embodiments, an antibody or conjugate of the present disclosure comprises a VH domain comprising the three CDRs of a single antibody shown in Table 4, and a VL domain comprising the three CDRs of a single antibody shown in Table 4. In some embodiments, an antibody or conjugate of the present disclosure comprises a VH domain comprising a CDR-H1 comprising the sequence of SEQ ID NO: 113, a CDR-H2 comprising the sequence of SEQ ID NO: 115, and a CDR-H3 comprising the sequence of SEQ ID NO: 116. In some embodiments, an antibody or conjugate of the present disclosure comprises a VH domain comprising a CDR-H1 comprising the sequence of SEQ ID NO: 114, a CDR-H2 comprising the sequence of SEQ ID NO: 189, and a CDR-H3 comprising the sequence of SEQ ID NO: 116. In some embodiments, the antibodies or conjugates of the present disclosure comprise a VL domain comprising a CDR-L1 comprising the sequence of SEQ ID NO: 117, a CDR-L2 comprising the sequence of SEQ ID NO: 119, and a CDR-L3 comprising the sequence of SEQ ID NO: 120. In some embodiments, the antibodies or conjugates of the present disclosure comprise a VL domain comprising a CDR-L1 comprising the sequence of SEQ ID NO: 118, a CDR-L2 comprising the sequence of SEQ ID NO: 177, and a CDR-L3 comprising the sequence of SEQ ID NO: 120. In some embodiments, the antibodies or conjugates of the present disclosure comprise a VL domain comprising a CDR-L1 comprising the sequence of SEQ ID NO: 117, a CDR-L2 comprising the sequence of SEQ ID NO: 119, and a CDR-L3 comprising the sequence of SEQ ID NO: 121.In some embodiments, an antibody or conjugate of the present disclosure comprises a VL domain comprising a CDR-L1 comprising the sequence of SEQ ID NO: 117, a CDR-L2 comprising the sequence of SEQ ID NO: 119, and a CDR-L3 comprising the sequence of SEQ ID NO: 122. In some embodiments, an antibody or conjugate of the present disclosure comprises a VH domain comprising a CDR-H1 comprising the sequence of SEQ ID NO: 113, a CDR-H2 comprising the sequence of SEQ ID NO: 115, and a CDR-H3 comprising the sequence of SEQ ID NO: 116, and a VL domain comprising a CDR-L1 comprising the sequence of SEQ ID NO: 117, a CDR-L2 comprising the sequence of SEQ ID NO: 119, and a CDR-L3 comprising the sequence of SEQ ID NO: 120. In some embodiments, the antibodies or conjugates of the present disclosure comprise a VH domain comprising a CDR-H1 comprising the sequence of SEQ ID NO: 113, a CDR-H2 comprising the sequence of SEQ ID NO: 115, and a CDR-H3 comprising the sequence of SEQ ID NO: 116, and a VL domain comprising a CDR-L1 comprising the sequence of SEQ ID NO: 117, a CDR-L2 comprising the sequence of SEQ ID NO: 119, and a CDR-L3 comprising the sequence of SEQ ID NO: 121. In some embodiments, the antibodies or conjugates of the present disclosure comprise a VH domain comprising a CDR-H1 comprising the sequence of SEQ ID NO: 113, a CDR-H2 comprising the sequence of SEQ ID NO: 115, and a CDR-H3 comprising the sequence of SEQ ID NO: 116, and a VL domain comprising a CDR-L1 comprising the sequence of SEQ ID NO: 117, a CDR-L2 comprising the sequence of SEQ ID NO: 119, and a CDR-L3 comprising the sequence of SEQ ID NO: 122.

[0219] Table 4. Anti-CD22 Antibody CDR Sequences

[0220]

[0221]

[0222] In certain embodiments, the anti-CD22 antibody is an antibody comprising a VH and a VL as shown in Table 5 below.

[0223] Table 5. Anti-CD22 antibody sequences

[0224]

[0225]

[0226] In some embodiments, the anti-CD22 antibody or conjugate comprises a VH domain shown in Table 6 and a VL domain shown in Table 7. For example, in some embodiments, the anti-CD22 antibody or conjugate comprises a VH domain comprising a sequence selected from the group consisting of SEQ ID NOs: 64-67 and a VL domain comprising a sequence selected from the group consisting of 68-91. In some embodiments, the anti-CD22 antibody or conjugate comprises a VH domain comprising the sequence of SEQ ID NO: 65 and a VL domain comprising the sequence of SEQ ID NOs: 73 or 87. In some embodiments, the anti-CD22 antibody or conjugate comprises a VH domain comprising the sequence of SEQ ID NO: 65 and a VL domain comprising the sequence of SEQ ID NO: 73. In some embodiments, the anti-CD22 antibody or conjugate comprises a VH domain comprising the sequence of SEQ ID NO: 65 and a VL domain comprising the sequence of SEQ ID NO: 87.

[0227] Table 6. Anti-CD22 antibody VH sequences.

[0228]

[0229] Table 7. Anti-CD22 antibody VL sequences.

[0230]

[0231]

[0232]

[0233] In some embodiments, the anti-CD22 antibody or conjugate comprises a heavy chain comprising a VH domain shown in Table 6 and a heavy chain constant domain sequence shown in Table 8A. In some embodiments, the anti-CD22 antibody or conjugate comprises a heavy chain comprising a VL domain shown in Table 7 and a light chain constant domain sequence shown in Table 8A.

[0234] Table 8A. Antibody constant domain sequences.

[0235]

[0236]

[0237]

[0238]

[0239]

[0240]

[0241] In some embodiments, the anti-CD22 antibody or conjugate comprises a heavy chain (including a Q tag) as shown in Table 8B and a light chain as shown in Table 8B. In some embodiments, the anti-CD22 antibody or conjugate comprises a heavy chain comprising the sequence of SEQ ID NO: 179 or 180 and a light chain comprising the sequence of SEQ ID NO: 181 or 182. In some embodiments, the anti-CD22 antibody or conjugate comprises a heavy chain comprising the sequence of SEQ ID NO: 179 and a light chain comprising the sequence of SEQ ID NO: 181. In some embodiments, the anti-CD22 antibody or conjugate comprises a heavy chain comprising the sequence of SEQ ID NO: 179 and a light chain comprising the sequence of SEQ ID NO: 182. In some embodiments, the anti-CD22 antibody or conjugate comprises a heavy chain comprising the sequence of SEQ ID NO: 180 and a light chain comprising the sequence of SEQ ID NO: 181. In some embodiments, the anti-CD22 antibody or conjugate comprises a heavy chain comprising the sequence of SEQ ID NO: 180 and a light chain comprising the sequence of SEQ ID NO: 182. In some embodiments, the anti-CD22 antibody or conjugate comprises two heavy chains and two light chains, each heavy chain comprising the sequence of SEQ ID NO: 179 or 180, and each light chain comprising the sequence of SEQ ID NO: 181 or 182. In some embodiments, the anti-CD22 antibody or conjugate comprises two heavy chains and two light chains, each heavy chain comprising the sequence of SEQ ID NO: 179, and each light chain comprising the sequence of SEQ ID NO: 181. In some embodiments, the anti-CD22 antibody or conjugate comprises two heavy chains and two light chains, each heavy chain comprising the sequence of SEQ ID NO: 179, and each light chain comprising the sequence of SEQ ID NO: 182. In some embodiments, the anti-CD22 antibody or conjugate comprises two heavy chains and two light chains, each heavy chain comprising the sequence of SEQ ID NO: 180, and each light chain comprising the sequence of SEQ ID NO: 181. In some embodiments, the anti-CD22 antibody or conjugate comprises two heavy chains and two light chains, each heavy chain comprising the sequence of SEQ ID NO: 180 and each light chain comprising the sequence of SEQ ID NO: 182.

[0242] Table 8B. Anti-CD22 antibody sequences (including C-terminal Q tag on the heavy chain)

[0243]

[0244] In some embodiments, a conjugate according to Table 11 (eg, Conjugate AF) is used in the methods of the present disclosure.

[0245] Table 11. Exemplary anti-CD22 antibody-CpG oligonucleotide conjugates.

[0246]

[0247] III.Medicine Box

[0248] Also provided herein are kits comprising a conjugate as described above, for use in any of the methods described herein.

[0249] In another aspect, the kit also includes a package insert, including but not limited to appropriate instructions for preparation and administration of the formulation, side effects of the formulation, and any other relevant information. The instructions may be in any suitable format, including but not limited to printed matter, videotape, computer readable disk, CD-ROM, or Internet-based instructions.

[0250] In another aspect, a kit for treating an individual suffering from or susceptible to a disorder described herein is provided, comprising a first container containing a dose of a composition or formulation described herein, and a package insert. The container can be any container known in the art and suitable for storing and delivering intravenous formulations. In certain embodiments, the kit further comprises a second container containing a pharmaceutically acceptable carrier, diluent, adjuvant, etc., for preparing a formulation to be administered to an individual.

[0251] In another aspect, kits containing sufficient doses of the compositions described herein (including pharmaceutical compositions thereof) to provide effective treatment to an individual over an extended period of time, such as 1-3 days, 1-5 days, one week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 1 cycle, 2 cycles, 3 cycles, 4 cycles, 5 cycles, 6 cycles, 7 cycles, 8 cycles or longer, may also be provided.

[0252] In some embodiments, kits may also include multiple doses and may be packaged in quantities sufficient for storage and use in pharmacies (e.g., hospital pharmacies and compounding pharmacies). In certain embodiments, kits may include a dose of at least one composition disclosed herein.

[0253] Example

[0254] The presently disclosed subject matter will be better understood with reference to the following examples, which are provided by way of illustration and not limitation of the invention.

[0255] Example 1: An open-label, dose-escalation study evaluating the safety, tolerability, pharmacokinetics, preliminary efficacy and pharmacokinetics of an anti-CD22-CpG conjugate in patients with advanced or metastatic solid tumors

[0256] The conjugate containing an anti-CD22 antibody (Ab) and an immunomodulatory oligonucleotide (also referred to as an "anti-CD22-CpG conjugate") used in this study is a Toll-like receptor (TLR) agonist antibody conjugate (TRAAC) designed to deliver TLR9 activation in targeted immune cells by systemic administration. The anti-CD22-CpG conjugate contains a TLR9 agonist oligonucleotide (14-mer) site that is specifically conjugated to the anti-CD22 antibody at the C-terminus via a Q-tag peptide described herein. Preclinical mouse tumor models have shown that anti-CD22-CpG conjugate treatment activates the TLR9 pathway in B cells to increase immune activation, cytokine production, antigen presentation, and anti-tumor efficacy. This study aims to test the safety, tolerability, pharmacokinetics, preliminary efficacy, and pharmacodynamics of anti-CD22-CpG conjugates for the treatment of patients with advanced or metastatic solid tumors.

[0257] In some embodiments, one of conjugates AF is administered, as shown in Table 11.

[0258] Anti-CD22-CpG conjugate C is administered to patients by intravenous infusion every 2 weeks (Q2W) for about 90 minutes, with a starting dose of 0.1 mg / kg. During the first week after receiving the first dose, the patient's daily body temperature and any CRS symptoms are monitored. Patients are monitored for 28 days to ensure that there is no dose-limiting toxicity (DLT), and the DLT target rate is 25%. During the 29th to 60th day, all patients were also monitored for late DLT. In the absence of any DLT or ≥2-grade cytokine release syndrome (CRS), the next group of patients will be dose-upgraded to the next dose of 0.3 mg / kg Q2W, and monitored for 28 days. This process continues, with each dose increasing by about 3 times (i.e., dose 1 = 0.1 mg / kg, dose 2 = 0.3 mg / kg, dose 3 = 1.0 mg / kg, dose 4 = 3.0 mg / kg). Additional increments are dose 5 = 10.0 mg / kg, dose 6 = 30.0 mg / kg, and optional dose 7 = maximum 60.0 mg / kg. If DLT or ≥ Grade 2 CRS is observed, the subsequent dose level is increased by up to 2 times (for example, if DLT is observed at dose 1 = 0.1 mg / kg, the maximum dose 2 = 0.2 mg / kg). Dose level 7 is optional, not exceeding 60 mg / kg, and depends on the available safety, pharmacokinetic and efficacy data collected from other dose groups. A Bayesian optimal interval (BOIN) design with a 3+3 design run is used to inform dose escalation / decrease decisions for dose levels. Up to 30 patients may be recruited to the dose escalation phase to determine the maximum tolerated dose (MTD). In order to confirm the most ideal dose for further evaluation or to determine the recommended phase 2 dose (RP2D), at least two dose optimization cohorts will be recruited with dose levels equal to or lower than the MTD. A total of 100 patients can be recruited in the dose escalation and dose optimization phases, initially recruiting up to 5 patients at a specific dose, with a 48-hour interval between the first patient and subsequent patients to assess whether there are any initial safety issues.

[0259] The study consisted of a screening period, a treatment period, and a follow-up period. All patients completed a screening period of up to 28 days. During the treatment period, patients received anti-CD22-CpG conjugate every two weeks (Q2W) until the treatment discontinuation criteria were met. Patients then entered the follow-up period, which will last up to 2 years or until the study discontinuation criteria were met.

[0260] Inclusion criteria included:

[0261] (a) Age (≥18 years);

[0262] (b) gender (all);

[0263] (c) Solid tumors documented as inoperable, locally advanced, metastatic, or recurrent cancers as documented by histology or cytology:

[0264] (i) Skin melanoma;

[0265] (ii) breast cancer;

[0266] (iii) ovarian cancer;

[0267] (iv) colorectal cancer;

[0268] (v) non-small cell lung cancer (NSCLC);

[0269] (vi) renal cell carcinoma (RCC);

[0270] (vii) Head and neck squamous cell carcinoma (HNSCC):

[0271] (viii) Merkel cell carcinoma;

[0272] (ix) hepatocellular carcinoma (HCC);

[0273] (x) cervical squamous cell carcinoma;

[0274] (xi) urothelial carcinoma;

[0275] (xii) Endometrial cancer

[0276] (xiii) gastroesophageal adenocarcinoma; and

[0277] (xiv) bile duct cancer;

[0278] (d) Patients must have at least one measurable lesion as defined by RECIST v1.1.

[0279] (e) Male or female adult patients aged ≥18 years on the date of signing the informed consent.

[0280] (f) Eastern Cooperative Oncology Group (ECOG) performance status (PS) of 0 or 1;

[0281] (g) Demonstration of adequate organ function.

[0282] Exclusion criteria include:

[0283] (a) In addition to the disease treated in this study, the patient has a history of other malignant diseases or has other active malignant diseases;

[0284] (b) known brain metastases or epidural disease;

[0285] (c) receiving chronic systemic steroid therapy (>10 mg / day prednisone or equivalent) or any immunosuppressive therapy within 7 days before the first dose of study drug;

[0286] (d) active autoimmune disease requiring systemic treatment in the past 2 years;

[0287] (e) a history of grade 3 or higher immune-mediated adverse events and previous immunotherapy that was considered to be drug-related;

[0288] (f) infection with HIV-1 or HIV-2, unless HIV is well controlled and the viral load is undetectable; infection with hepatitis B, unless the disease is undetectable; infection with hepatitis C, unless the disease is well controlled and the viral load is minimal;

[0289] (g) active infection requiring systemic treatment;

[0290] (h) suffering from severe cardiovascular disease in the past 6 months;

[0291] (i) Receiving any type of systemic anticancer therapy within 4 weeks after the first dose of study drug;

[0292] (j) Currently or previously participated in an interventional clinical trial using an investigational compound or device within 4 weeks of first administration of the investigational drug;

[0293] (k) received radiation therapy for bone metastasis within 2 weeks after the first dose of study drug, or received any other radiation therapy within 4 weeks;

[0294] (l) Underwent major surgery within 2 weeks after first taking the study drug;

[0295] (m) any live vaccine within 4 weeks or any other vaccine within 2 days after the first dose of study drug;

[0296] (n) any previous treatment with TLR9 agonists or anti-CD22 antibodies;

[0297] (o) Known allergy to components of the study therapeutic drug or its analogs;

[0298] (p) any historical or current evidence of treatment for any condition, laboratory abnormality, or other condition that would place the subject at risk for participation in the trial, confound the trial results, or interfere with the patient's participation throughout the trial; and

[0299] (q) A mental or substance abuse disorder known to interfere with compliance with the test requirements.

[0300] The primary outcome measures included safety and tolerability analysis of increasing dose levels of the anti-CD22-CpG conjugate to confirm the recommended dose for the Phase 2 extension study. This outcome measure included assessment of 28-day dose-limiting toxicity (DLT), adverse events (AEs), and laboratory abnormalities. AEs were characterized by type, frequency, and severity according to the National Cancer Institute Common Terminology Criteria for Adverse Events (NCICTCAE) v 5.0, and laboratory abnormalities were graded according to CTCAE v 5.0.

[0301] Secondary outcome measures included the following:

[0302] (a) Single-dose and multiple-dose pharmacokinetic characterization of anti-CD22-CpG conjugates, including pharmacokinetic parameters such as serum concentration (C max ), the shortest time to reach peak plasma concentration (T max ), area under the curve (AUC), drug clearance (CL) and half-life (t 1 / 2 ) (if the data permits);

[0303] (b) evaluating the immunogenicity of the anti-CD22-CpG conjugate and the incidence of anti-drug antibodies against the anti-CD22-CpG conjugate; and

[0304] (c) Preliminary antitumor activity was evaluated for all groups according to best overall response rate (ORR) and duration of response (DOR) and clinical benefit rate according to RECIST v1.1 and iRECIST, and for patients with ovarian cancer according to RECIST v1.1 and Gynecologic Cancer Intergroup (GCIG) CA-125 ORR and DOR criteria. The response criteria are shown in Tables E1-E2C below.

[0305] Exploratory outcome measures included the following:

[0306] (a) Characterization of other efficacy outcomes, such as progression-free survival (PFS) and overall survival (OS);

[0307] (b) characterizing the extent of target engagement of the anti-CD22-CpG conjugate by measuring CD22 expression on B cells in peripheral blood;

[0308] (c) exploring the effects of anti-CD22-CpG conjugates on systemic soluble immune factors, including quantification of soluble immune factors in serum before and after administration;

[0309] (d) evaluating the pharmacodynamic effects of anti-CD22-CpG conjugates on immune cell markers, including the levels of phenotypic markers on B cells and lymphocyte counts in peripheral blood; and

[0310] (e) exploring the effects of anti-CD22-CpG conjugates on pharmacodynamic markers in tumor tissues, including the levels of pharmacodynamic markers before and after administration, including but not limited to (i) tumor mutation burden (TMB) and (ii) PD-L1 and other immunomodulatory markers.

[0311] Table E1. Response criteria for evaluation of target lesions.

[0312]

[0313] 1 When lymph node lesions are part of the target lesion, the SOD of CR may not be zero

[0314] 2 After an initial CR, if all non-nodal target lesions remain absent and all nodal lesions are <10 mm in size, they cannot be assigned PD. In this case, the target lesion response is a CR.

[0315] Table E2A. Assessment of overall cancer response. See Table E1 for abbreviations.

[0316]

[0317]

[0318] 3 Does not meet criteria for CR or PD.

[0319] All significant regressions should be confirmed ≥4 weeks after response criteria were first met or at the next tumor assessment.

[0320] Table E2B. Assignment of time point responses using iRECIST. See Table E1 for abbreviations.

[0321]

[0322]

[0323] 4 It had been determined in the assessment prior to this time point. “i” indicates immune response assigned using iRECIST.

[0324] Abbreviations: iCR = complete response; iPR = partial response; iSD = stable disease; iUPD = unconfirmed progressive disease; non-iCR / non-iPD = CR or PD criteria not met; iCPD = confirmed progressive disease; RECIST = response evaluation criteria in solid tumors.

[0325] Table E2C. Overall response in patients with initial measurable disease and evaluable by CA-125, combined with CA-125 and RECIST criteria. For abbreviations, see Table E1.

[0326]

[0327] 5 Target lesions included up to 5 measurable lesions defined by RECIST.

[0328] 6 Nontarget lesions included ascites and peritoneal thickening that were not measurable according to RECIST.

[0329] 7 Patients with a CA-125 response more than 28 days from PD according to RECIST were considered PR based on best response, but were considered PD if RECIST PD was within 28 days of CA-125 response.

[0330] Best RECIST response also needed to be confirmed and maintained for at least 28 days.

[0331] Example 2: CD22 target engagement

[0332] The ability of the administered anti-CD22-CpG conjugate to express and maintain CD22 target (CD22) engagement was evaluated. Using the regimen of Example 1, anti-CD22-CpG conjugate C was administered to patients at 0.1 mg / kg (1 patient), 0.3 mg / kg (1 patient), 1 mg / kg (7 patients) or 3 mg / kg (4 patients) in about 90 minutes by intravenous infusion once every two weeks (Q2W). Peripheral whole blood (PB) was drawn before the first dose on day 1 (C1D1 pre) and 3 hours after administration (C1D1 3hrs) in cycle 1, and then on day 2 (C1D2), day 8 (C1D8), day 15 before the second administration (C1D15 pre), and blood was drawn at time points in subsequent cycles.

[0333] CD19 from PB was analyzed by fluorescence activated cell sorting (FACS) analysis using a Navios flow cytometer (Beckman Coulter) or a CytoFLEX LX cytometer (Beckman Coulter). + CD22 surface expression on B cells. Data analysis was performed using WinList (Verity software) or FlowLogic software. B cells were identified as CD45 + CD19 +Lymphocytes. The equivalent soluble fluorescent dye molecules (MESF) values ​​of cell surface CD22 expression were determined using SpheroTech Rainbow calibration beads URCP-38-2K (SpheroTech) according to the manufacturer's recommendations. Data were tabulated using GraphPad Prism software. Data are expressed as the mean percentage change in CD22 cell surface expression relative to pre-dose values ​​+ / - standard deviation (SD).

[0334] like Figure 2A As shown, the expression level of cell surface CD22 on peripheral B cells decreased after administration of anti-CD22-CpG conjugates. Higher doses tended to prolong target engagement, as shown by the slower rate at which cell surface CD22 returned to pre-dose levels. Patients at the 0.3 mg / kg and 1 mg / kg doses were re-evaluated after the second administration cycle. Figure 2B The anti-CD22-CpG conjugates at dose levels of 0.3 mg / kg and 1 mg / kg demonstrated similar levels of target engagement at doses 1 and 4. At the higher dose level of 3 mg / kg, anti-CD22-CpG conjugates, the data indicated that the conjugates maintained target engagement prior to and during the second treatment cycle (4 doses; 2 cycles of twice weekly administration).

[0335] Example 3: B cell activation

[0336] B cell activation in response to administration of anti-CD22-CpG conjugates was assessed. Activation of B cells in PB collected from patients in Example 2 was analyzed by measuring CD86 expression using a Navios flow cytometer (Beckman Coulter) or a CytoFLEX LX cytometer (Beckman Coulter) by FACS analysis. Data analysis was performed using WinList (Verity Software) or FlowLogic Software. Naive B cells were identified as CD45 + CD19 + CD27 - , memory B cells were identified as CD45 + CD19 + CD27 + CD86 expression was derived by gating on the percentage of naive and memory B cells expressing CD86. Data were tabulated using GraphPad Prism software and expressed as fold change from pre-dose marker expression.

[0337] like Figure 3A and 3B As shown, all doses of anti-CD22-CpG conjugates caused B cell activation as evidenced by increased CD86 expression. Figure 3C Evidence for B cell activation after the second administration cycle (4 doses; 2 cycles of biweekly administration) is provided.

[0338] Example 4: Pharmacokinetics

[0339] The pharmacokinetics of the anti-CD22-CpG conjugate was evaluated by measuring the concentration of the conjugate in patients administered 0.1, 0.3 and 1 mg / kg of Example 2. The conjugate was detected using the MesoScale Discoveries (MSD) assay using the human CD22 ECD (TNT9, Tallac Therapeutics) ligand-bound format and electrochemiluminescence (ECL) technology. The conjugate was detected using biotin-conjugated antisense oligonucleotides (primary detection agents) and Sulfo-TAG-labeled streptavidin. After adding read buffer T, the electrochemiluminescence signal was read on an MSD Sector Imager plate reader. The results are shown in Tables E4 and Figure 4 Available in.

[0340] Table E3. Pharmacokinetics of anti-CD22-CpG conjugates

[0341]

[0342] Cmax at 1 mg / kg is expressed as mean (SD)

[0343] Between 0.1 and 1 mg / kg doses, the C max The increase was roughly proportional to the dose. For the 0.1 and 1 mg / kg doses, the increase in AUC was greater than dose proportional. A similar trend was observed when the total antibody of the samples was measured.

[0344] At the time of filing this application, three of the eight evaluable subjects demonstrated stable disease according to the criteria in Example 1. The dose levels for these subjects were 0.3, 1, and 3 mg / kg, respectively.

Claims

1. A method of treating cancer in an individual, comprising administering to the individual a conjugate comprising an anti-CD22 antibody (Ab) and an immunomodulatory oligonucleotide (P) at a dose of 0.1 mg / kg to 60 mg / kg; Wherein the Ab comprises two antibody light chains, two antibody heavy chains and two Q tag peptides (Q); wherein each of the two Q tag peptides comprises the amino acid sequence RPQGFGPP (SEQ ID NO: 49); wherein one of the Q tag peptides is connected to the C-terminus of each of the two antibody heavy chains; wherein one of the two Q tag peptides is connected to the immunomodulatory oligonucleotide via an amide bond with a glutamine residue of the Q tag peptide and a linker (L), as shown in formula (A): in represents the connection point between Q and the antibody (Ab); wherein each heavy chain of the Ab comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 113, a CDR2 comprising an amino acid sequence of SEQ ID NO: 115, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 116, and each light chain of the Ab comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 117, a CDR2 comprising an amino acid sequence of SEQ ID NO: 119, and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 120-122; Wherein the linker L is Where m is 24, and where represents the point of attachment to the oligonucleotide P, and represents the point of attachment to the glutamine residue of the Q tag; and Wherein the oligonucleotide P comprises the following structure: or a pharmaceutically acceptable salt thereof, in *and ** indicates a point of attachment within the oligonucleotide, wherein Indicates the connection point with the linker L.

2. The method of claim 1, wherein the individual has an inoperable, locally advanced, metastatic and / or recurrent solid tumor.

3. The method of claim 1 or claim 2, wherein the individual has a cancer selected from the group consisting of: cutaneous melanoma, breast cancer, ovarian cancer, colorectal cancer, non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC), head and neck squamous cell carcinoma (HNSCC), Merkel cell carcinoma, hepatocellular carcinoma (HCC), cervical squamous cell carcinoma, urothelial carcinoma, endometrial carcinoma, gastroesophageal adenocarcinoma, and bile duct cancer.

4. A method of treating cancer in an individual, comprising administering to the individual a conjugate comprising an anti-CD22 antibody (Ab) and an immunomodulatory oligonucleotide (P); wherein the individual has an inoperable, locally advanced, metastatic and / or recurrent solid tumor; Wherein the Ab comprises two antibody light chains, two antibody heavy chains and two Q tag peptides (Q); wherein each of the two Q tag peptides comprises the amino acid sequence RPQGFGPP (SEQ ID NO: 49); wherein one of the Q tag peptides is connected to the C-terminus of each of the two antibody heavy chains; wherein one of the two Q tag peptides is connected to the immunomodulatory oligonucleotide via an amide bond with a glutamine residue of the Q tag peptide and a linker (L), as shown in formula (A): in represents the connection point between Q and the antibody (Ab); wherein each heavy chain of the Ab comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 113, a CDR2 comprising an amino acid sequence of SEQ ID NO: 115, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 116, and each light chain of the Ab comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 117, a CDR2 comprising an amino acid sequence of SEQ ID NO: 119, and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 120-122; Wherein the linker L is Where m is 24, and where represents the point of attachment to the oligonucleotide P, and represents the point of attachment to the glutamine residue of the Q tag; and Wherein the oligonucleotide P comprises the following structure: or or a pharmaceutically acceptable salt thereof, in *and ** indicates a point of attachment within the oligonucleotide, and wherein Indicates the connection point with the linker L.

5. The method of claim 4, wherein the individual has a cancer selected from the group consisting of: cutaneous melanoma, breast cancer, ovarian cancer, colorectal cancer, non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC), head and neck squamous cell carcinoma (HNSCC), Merkel cell carcinoma, hepatocellular carcinoma (HCC), cervical squamous cell carcinoma, urothelial carcinoma, endometrial carcinoma, gastroesophageal adenocarcinoma, and bile duct cancer.

6. The method of claim 4 or claim 5, wherein the conjugate is administered to the individual at a dose of between 0.1 mg / kg and 60 mg / kg.

7. The method of any one of claims 1-6, wherein the individual has a solid tumor that has progressed on and / or is intolerant to standard therapy.

8. The method of any one of claims 1-7, wherein the conjugate is administered to the individual at a dose of 0.1, 0.3, 1.0, 3.0, 10.0, 30.0 or 60.0 mg / kg.

9. The method of any one of claims 1-8, wherein the conjugate is administered to the individual once every two weeks.

10. The method of any one of claims 1 to 9, wherein each heavy chain of the Ab comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 113, a CDR2 comprising the amino acid sequence of SEQ ID NO: 115, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 116, and each light chain of the Ab comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 117, a CDR2 comprising the amino acid sequence of SEQ ID NO: 119, and a CDR3 comprising the amino acid sequence of SEQ ID NO:

120.

11. The method of any one of claims 1-9, wherein each heavy chain of the Ab comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 113, a CDR2 comprising the amino acid sequence of SEQ ID NO: 115, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 116, and each light chain of the Ab comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 117, a CDR2 comprising the amino acid sequence of SEQ ID NO: 119, and a CDR3 comprising the amino acid sequence of SEQ ID NO:

121.

12. The method of any one of claims 1-9, wherein each heavy chain of the Ab comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 113, a CDR2 comprising the amino acid sequence of SEQ ID NO: 115, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 116, and each light chain of the Ab comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 117, a CDR2 comprising the amino acid sequence of SEQ ID NO: 119, and a CDR3 comprising the amino acid sequence of SEQ ID NO:

122.

13. The method of any one of claims 1-9, wherein each heavy chain of the Ab comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:

65.

14. The method of any one of claims 1-9 and 13, wherein each light chain of the Ab comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:

73.

15. The method of any one of claims 1-9 and 13, wherein each light chain of the Ab comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:

87.

16. The method of any one of claims 1-9, wherein each heavy chain of the Ab comprising the Q tag peptide comprises the amino acid sequence of SEQ ID NO: 179 or 180; and wherein each light chain of the Ab comprises the amino acid sequence of SEQ ID NO: 181 or 182.

17. The method of claim 16, wherein each heavy chain of the Ab comprising the Q-tag peptide comprises the amino acid sequence of SEQ ID NO: 179; and wherein each light chain of the Ab comprises the amino acid sequence of SEQ ID NO:

181.

18. The method of claim 16, wherein each heavy chain of the Ab comprising the Q-tag peptide comprises the amino acid sequence of SEQ ID NO: 179; and wherein each light chain of the Ab comprises the amino acid sequence of SEQ ID NO:

182.

19. The method of claim 16, wherein each heavy chain of the Ab comprising the Q-tag peptide comprises the amino acid sequence of SEQ ID NO: 180; and wherein each light chain of the Ab comprises the amino acid sequence of SEQ ID NO:

181.

20. The method of claim 16, wherein each heavy chain of the Ab comprising the Q-tag peptide comprises the amino acid sequence of SEQ ID NO: 180; and wherein each light chain of the Ab comprises the amino acid sequence of SEQ ID NO:

182.

21. The method of any one of claims 1-20, wherein the individual is a human.

22. The method of any one of claims 1-21, wherein the conjugate is administered in a formulation comprising: (a) conjugate at a concentration of 30 mg / mL; (b) 20 mM citrate; (c) 150 mM L-arginine; (d) 50 mM NaCl; and (e) 0.02% polysorbate 80 (w / v); The pH of the preparation is 6.

5.

23. The method of any one of claims 1-22, wherein the conjugate is administered to the individual intravenously.

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