Manipulated bispecific molecules and methods of use

Abstract

Provided herein are a bispecific molecule comprising a first domain and a second domain, and a method of treatment using the bispecific molecule, wherein the first domain binds to TREM1 or a functional fragment thereof, and the second domain binds to IL-17, IL-17R, or a functional fragment thereof. The present invention provides, for example, a bispecific molecule comprising a first domain and a second domain, wherein the first domain binds to TREM1 or a functional fragment thereof, and the second domain binds to IL-17, IL-17R, or a functional fragment thereof.

Description

Technical Field

[0001] Cross-Referenced Applications This application claims the benefit of priority based on U.S. Patent Provisional Application No. 63 / 509,594, filed on Jun. 22, 2023, the entire content of which is incorporated herein by reference.

[0002] Incorporation by Reference of Sequence Listing This application contains a sequence listing submitted via Patent Center. The sequence listing titled 220710-701601_PCT_SL.xml, created on Jun. 21, 2024, is 667,000 bytes in size and the entire content thereof is incorporated herein by reference.

[0003] Field The present disclosure generally relates to bispecific molecules that bind to TREM1 and interleukin.

Background Art

[0004] Background of the Present Disclosure A bispecific antibody (BsAb) is an antibody that has two binding sites that independently target two different antigens or two different epitopes on the same antigen. The therapeutic use of BsAb has been shown to offer the potential for enhanced activity compared to that of monospecific antibodies. BsAb is understood to have a wider use for immunotherapy in the treatment of various diseases.

Summary of the Invention

Means for Solving the Problems

[0005] Gist of the Present Disclosure Multispecific antibodies and other exemplary compositions that bind to both TREM1 and interleukins are provided. For example, in some embodiments, a multispecific antibody, such as a bispecific antibody, binds to TREM1 and a member of the IL17 family of interleukins, such as at least one of IL-17A, IL-17B, IL-17C, IL-17D, IL17-E, and IL-17F. Alternatively, the bispecific antibody binds to an interleukin receptor such as IL-17R, or to a complex of an interleukin receptor with its corresponding interleukin. This bibinding specificity can be achieved by manipulating an antibody having two different antigen-binding domains, one of which binds to TREM1 and the other to an interleukin. The compositions disclosed herein, but are not limited to, are useful for treating a variety of conditions in human subjects, including rheumatoid arthritis, juvenile arthritis, psoriatic arthritis, ankylosing spondylitis, axial spondyloarthritis, psoriasis, sweat gland abscess, ulcerative colitis, Crohn's disease, necrotizing enterocolitis, sepsis, or multiple sclerosis.

[0006] For example, a bispecific molecule comprising a first domain and a second domain is disclosed herein, wherein the first domain binds to TREM1 or a functional fragment thereof, and the second domain binds to an interleukin or a functional fragment thereof. In some embodiments, the bispecific molecule is an antibody, a variant of an antibody, or an engineered functional fragment of an antibody. In some embodiments, the second binding domain binds to an interleukin, which is IL-17, or a functional fragment thereof. In some embodiments, the bispecific molecule described herein binds to TREM1 in a soluble form, i.e., sTREM1. In some embodiments, the bispecific molecule described herein binds to a region of an interleukin, which is IL-17. In some embodiments, IL-17 includes IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, IL-17F, a dimer form, e.g., IL17A / F, or any combination thereof. In certain embodiments, IL-17 is IL-17A, IL-17F, a heterodimer of IL-17A and IL-17F, or any combination thereof. In some embodiments, the binding affinity of the first domain to TREM1 is lower than the binding affinity of the second domain to IL-17, IL-17R, or a functional fragment thereof. In some embodiments, the binding affinity of the second domain to IL-17, IL-17R, or a functional fragment thereof is at least twice the binding affinity of the first domain to TREM1 and lower than the binding affinity of the second domain to IL-17, IL-17R, or a functional fragment thereof. Alternatively, in some embodiments, the binding affinity of the first domain to TREM1 is higher than the binding affinity of the second domain to IL-17, IL-17R, or a functional fragment thereof. In some embodiments, the binding affinity of the first domain to TREM1 is at least twice the binding affinity of the second binding domain to IL-17, IL-17R, or a functional fragment thereof.

[0007] In some embodiments, the bispecific molecule described herein is a bispecific IgG antibody. In some embodiments, the bispecific molecule described herein is a Fab2 antibody, a bis-scFv antibody, a diabody, DVD-Ig, TandAb, a tandem scFv-Fc, a one-arm tandem scFv-Fc, DART, DART-Fc, or a functional fragment thereof. In some embodiments, the bispecific molecule comprises a heterodimer antibody, or its functional fragment comprises a constant region. In some embodiments, the molecule comprises modifications and / or sequence knockouts that enhance or reduce the polymerization of individual polypeptide chains. In some embodiments, the first domain comprises a TREM1-binding heavy chain variable domain. In some embodiments, the first domain comprises a TREM1-binding light chain variable domain. In some embodiments, the second domain comprises an IL-17-binding heavy chain variable domain. In some embodiments, the second domain comprises an IL-17-binding light chain variable domain. In some embodiments, at least one of the first and second domains includes a light chain constant domain and / or a heavy chain constant domain. In some embodiments, the bispecific molecule includes at least one of an Fc region and / or a Fab region. In some embodiments, the Fc region includes an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to any one of the amino acid sequences of SEQ ID NOs. 199, 722, and 723. In some embodiments, the heavy chain constant domain of the first domain includes an Fc region having S354C mutations and T366W mutations in EU numbering, and the heavy chain constant domain of the second domain includes an Fc region having Y349C mutations, T366S mutations, and Y407V mutations in EU numbering. In some embodiments, the heavy chain constant domain of the second domain includes an Fc region having S354C mutations and T366W mutations in EU numbering, and the heavy chain constant domain of the first domain includes an Fc region having Y349C mutations, T366S mutations and Y407V mutations in EU numbering.In some embodiments, the Fc region comprises a human IgG1 heavy chain constant having at least one substitution selected from the following EU numbering positions: N297, C226, C229, E233, L234, L235, G236, G237, P238, F243, M252, S254, T256, D265, S267, H268, D270, P271, R292, Y300, K322, A327, L328, P329, A330, P331, and P396. In some embodiments, the Fc region includes a human IgG double-chain constant chain having at least one substitution selected from positions C232, C233, V234, G237, P238, M252, S254, T256, H268, N297, V309, A330, and P331 in EU numbering. In some embodiments, the Fc region includes a human IgG quadruple-chain constant chain having at least one substitution selected from positions S228, E233, F234, L235, L236, G237, S241, L248, M252, S254, T256, N297, E318, and T394 in EU numbering. In some embodiments, the bispecific molecule contains at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, or higher anti-inflammatory activity compared to the anti-inflammatory activity of a combination of a monospecific antibody that binds to TREM1 and a monospecific antibody that binds to a cytokine of the IL-17 family, IL-17R, or a combination thereof.

[0008] Also disclosed herein are multispecific molecules for use in the treatment of inflammatory diseases or conditions. In some embodiments, the inflammatory disease or condition is selected from the group consisting of rheumatoid arthritis, juvenile arthritis, psoriatic arthritis, axial spondyloarthritis, or ankylosing spondylitis. In some embodiments, the bispecific molecule is for use in the treatment of psoriasis or sweat gland abscesses. In some embodiments, the bispecific molecule is for use in the treatment of ulcerative colitis, Crohn's disease, necrotizing enterocolitis, sepsis, or multiple sclerosis.

[0009] Furthermore, compositions comprising the bispecific molecules described herein are also disclosed herein. In some embodiments, the composition comprises a bispecific molecule comprising a first domain and a second domain, the first domain binding to TREM1 or a functional fragment thereof, and the second domain binding to IL-17 or a functional fragment thereof, or IL-17R or a functional fragment thereof, wherein administration of an effective amount of the composition to a subject requiring it results in treatment of an inflammatory disease or condition. In some embodiments, IL-17 comprises IL-17A, IL-17A / F, or any combination thereof. In some embodiments, the inflammatory disease or condition is selected from the group consisting of rheumatoid arthritis, juvenile arthritis, psoriatic arthritis, ankylosing spondylitis, axial spondylitis, psoriasis, sweat gland abscess, ulcerative colitis, Crohn's disease, necrotizing enterocolitis, sepsis, or multiple sclerosis. In some embodiments, the inflammatory disease or condition is rheumatoid arthritis, juvenile arthritis, psoriatic arthritis, axial spondylitis, or ankylosing spondylitis. In some embodiments, the inflammatory disease or condition is psoriasis or sweat gland abscess. In some embodiments, the inflammatory disease or condition is ulcerative colitis, Crohn's disease, necrotizing enterocolitis, sepsis, or multiple sclerosis. In some embodiments, the inflammatory disease or condition is sepsis. In some embodiments, the inflammatory disease or condition is multiple sclerosis.

[0010] Also disclosed herein are a pharmaceutical composition comprising the bispecific molecules and pharmaceutically acceptable carriers described herein, as well as a method for treating an inflammatory disease or condition in a subject by administering an effective amount of the multispecific molecules or pharmaceutical compositions disclosed herein to the subject.

[0011] Also disclosed herein are methods for treating autoimmune diseases in subjects, comprising administering an effective amount of a bispecific molecule to the subject, comprising a TREM1-binding domain containing at least one of the heavy chain complementarity-determining regions (CDRs) or light chain CDRs listed in Table 8, a TREM1-binding variant thereof, and an interleukin containing at least one of the heavy chain complementarity-determining regions (CDRs) or light chain CDRs listed in Table 8, or an IL-17-binding variant thereof. In some embodiments, the method involves nicotinamide phosphoribosyltransferase (NAMPT), dehydrogenase / reductase 9 (DHRS9), cyclin-dependent kinase inhibitor 1A (CDKN1A), CD52 molecule (CD52), myotubularin-related protein 11 (MTMR11), EH domain-containing protein 1 (EHD1), solute transporter family 27 member 3 (SLC27A3), interleukin 24 (IL24), Pim-2 oncogene serine / threonine kinase (PIM2), chitinase 3-like protein 1 (CHI3L1), polypeptide N-acetylgalactosaminyltransferase 6 (GALNT6), acyl-CoA thioesterase 7 (ACOT7), cytokine-inducible SH2-containing protein (CISH), sequence similarity family including member A (FAM129A), polo-like kinase 3 (PLK3), and major facilitator (major The method increases the expression of at least one of the following: facilitator) superfamily domain-containing 12 (MFSD12), StAR-related lipid transfer domain-containing 4 (STARD4), type c lectin domain family 12 member A (CLEC12A), CD55 molecule (Cromer blood group) (CD55), and interferon lambda receptor 1 (IFNLR1). In some embodiments, the method repairs the pentose phosphate pathway (PPP).

[0012] Also disclosed herein are methods for treating an inflammatory disease or condition in a subject, comprising administering an effective amount of the bispecific molecules, compositions, or pharmaceutical compositions described herein to the subject, thereby treating the inflammatory disease or condition. In some embodiments, the inflammatory disease or condition is associated with increased activity and / or expression of TREM1, IL-17 family cytokines, IL-17R, one or more downstream inflammatory signaling proteins, or combinations thereof, compared to the activity and / or expression of TREM1, IL-17 family cytokines, IL-17R, one or more downstream inflammatory signaling proteins, or combinations thereof, in a subject without an inflammatory disease or condition. In some embodiments, the method involves nicotinamide phosphoribosyltransferase (NAMPT), dehydrogenase / reductase 9 (DHRS9), cyclin-dependent kinase inhibitor 1A (CDKN1A), CD52 molecule (CD52), myotubularin-related protein 11 (MTMR11), EH domain-containing protein 1 (EHD1), solute transporter family 27 member 3 (SLC27A3), interleukin 24 (IL24), Pim-2 oncogene serine / threonine kinase (PIM2), chitinase 3-like protein 1 (CHI3L1), polypeptide N-acetylgalactosaminyltransferase 6. The method increases the expression of at least one of the following: (GALNT6), acyl-CoA thioesterase 7 (ACOT7), cytokine-inducible SH2-containing protein (CISH), family containing sequence similarity 129 member A (FAM129A), polo-like kinase 3 (PLK3), major facilitator superfamily domain-containing 12 (MFSD12), StAR-related lipid transfer domain-containing 4 (STARD4), c-type lectin domain family 12 member A (CLEC12A), CD55 molecule (Cromer blood group) (CD55), and interferon lambda receptor 1 (IFNLR1). In some embodiments, the method repairs the pentose phosphate pathway (PPP).

[0013] Also disclosed herein are methods for treating psoriasis in a subject, comprising administering an effective amount of a bispecific molecule, a composition, or a pharmaceutical composition described herein to the subject, thereby treating psoriasis. In some embodiments, the method reduces the incidence of Candida infection in a subject compared to a subject treated with a monospecific antibody that reduces IL-17 activity. In some embodiments, the method involves nicotinamide phosphoribosyltransferase (NAMPT), dehydrogenase / reductase 9 (DHRS9), cyclin-dependent kinase inhibitor 1A (CDKN1A), CD52 molecule (CD52), myotubularin-related protein 11 (MTMR11), EH domain-containing protein 1 (EHD1), solute transporter family 27 member 3 (SLC27A3), interleukin 24 (IL24), Pim-2 oncogene serine / threonine kinase (PIM2), chitinase 3-like protein 1 (CHI3L1), polypeptide N-acetylgalactosaminyltransferase 6 The method increases the expression of at least one of the following: (GALNT6), acyl-CoA thioesterase 7 (ACOT7), cytokine-inducible SH2-containing protein (CISH), family containing sequence similarity 129 member A (FAM129A), polo-like kinase 3 (PLK3), major facilitator superfamily domain-containing 12 (MFSD12), StAR-related lipid transfer domain-containing 4 (STARD4), c-type lectin domain family 12 member A (CLEC12A), CD55 molecule (Cromer blood group) (CD55), and interferon lambda receptor 1 (IFNLR1). In some embodiments, the method repairs the pentose phosphate pathway (PPP).

[0014] Also disclosed herein is a method for reducing an IL-17-related inflammatory state (or symptoms), comprising administering an effective amount of the bispecific molecule, composition, or pharmaceutical composition described herein to a subject, thereby reducing the IL-17-related inflammatory state (or symptoms) in the subject compared to the IL-17-related inflammatory state (or symptoms) in the subject before administration of the bispecific molecule. In some embodiments, the method involves nicotinamide phosphoribosyltransferase (NAMPT), dehydrogenase / reductase 9 (DHRS9), cyclin-dependent kinase inhibitor 1A (CDKN1A), CD52 molecule (CD52), myotubularin-related protein 11 (MTMR11), EH domain-containing protein 1 (EHD1), solute transporter family 27 member 3 (SLC27A3), interleukin 24 (IL24), Pim-2 oncogene serine / threonine kinase (PIM2), chitinase 3-like protein 1 (CHI3L1), polypeptide N-acetylgalactosaminyltransferase 6. The method increases the expression of at least one of the following: (GALNT6), acyl-CoA thioesterase 7 (ACOT7), cytokine-inducible SH2-containing protein (CISH), family containing sequence similarity 129 member A (FAM129A), polo-like kinase 3 (PLK3), major facilitator superfamily domain-containing 12 (MFSD12), StAR-related lipid transfer domain-containing 4 (STARD4), c-type lectin domain family 12 member A (CLEC12A), CD55 molecule (Cromer blood group) (CD55), and interferon lambda receptor 1 (IFNLR1). In some embodiments, the method repairs the pentose phosphate pathway (PPP).

[0015] Also disclosed herein is a method for reducing TREM1-related inflammatory conditions (or symptoms), comprising administering an effective amount of the bispecific molecule, composition, or pharmaceutical composition described herein to a subject, thereby reducing the TREM1-related inflammatory condition (or symptoms) in the subject compared to the TREM1-related inflammatory condition (or symptoms) in the subject before administration of the bispecific molecule. In some embodiments, the method involves nicotinamide phosphoribosyltransferase (NAMPT), dehydrogenase / reductase 9 (DHRS9), cyclin-dependent kinase inhibitor 1A (CDKN1A), CD52 molecule (CD52), myotubularin-related protein 11 (MTMR11), EH domain-containing protein 1 (EHD1), solute transporter family 27 member 3 (SLC27A3), interleukin 24 (IL24), Pim-2 oncogene serine / threonine kinase (PIM2), chitinase 3-like protein 1 (CHI3L1), polypeptide N-acetylgalactosaminyltransferase 6. The method increases the expression of at least one of the following: (GALNT6), acyl-CoA thioesterase 7 (ACOT7), cytokine-inducible SH2-containing protein (CISH), family containing sequence similarity 129 member A (FAM129A), polo-like kinase 3 (PLK3), major facilitator superfamily domain-containing 12 (MFSD12), StAR-related lipid transfer domain-containing 4 (STARD4), c-type lectin domain family 12 member A (CLEC12A), CD55 molecule (Cromer blood group) (CD55), and interferon lambda receptor 1 (IFNLR1). In some embodiments, the method repairs the pentose phosphate pathway (PPP).

[0016] Also disclosed herein are compositions comprising a bispecific molecule comprising a first domain and a second domain, wherein the first domain binds to TREM1 or a functional fragment thereof, and the second domain binds to an interleukin or a functional fragment thereof, and administration of an effective amount of the composition to a subject requiring it results in the treatment of an inflammatory disease or condition. In some embodiments, the interleukin comprises IL-17. In some cases, IL-17 is IL-17A, IL-17F, IL-17A / F, or any combination thereof. In some embodiments, the bispecific molecule is a Fab2 antibody, a bis-scFv antibody, a diabody, DVD-Ig, TandAb, a tandem scFv-Fc, a one-arm tandem scFv-Fc, DART, DART-Fc, or a functional fragment thereof. In some embodiments, the interleukin is IL-17 or a functional fragment thereof. In some embodiments, the bispecific molecule comprises a heterodimer antibody or a functional fragment thereof. In some embodiments, the bispecific molecule comprises a constant region. In some embodiments, the bispecific molecules include a sequence knockout in the constant region. Nucleic acids encoding at least a portion of any one of the bispecific molecules described herein are also disclosed herein. Embedding by reference

[0017] All publications, patents, and patent applications described herein are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application were specifically and individually incorporated by reference.

[0018] The features of this disclosure are described in particular in conjunction with the appended claims. A better understanding of the features and advantages of this disclosure can be obtained by referring to the following detailed description, which describes exemplary embodiments utilizing the principles of this disclosure, and to the appended drawings. [Brief explanation of the drawing]

[0019] [Figure 1] Figure 1 depicts a bispecific antibody containing both a TREM1-binding domain and an IL-17-binding domain.

[0020] [Figure 2] Figures 2A - 2B show the effect of contacting human peripheral blood mononuclear cells (PBMCs) with the bispecific antibodies described herein that target TREM1 and IL - 17. Briefly, Figure 2A shows the amount of IL - 17 present in the supernatant of PBMCs after treatment with the bispecific antibodies described herein. Figure 2B shows the amount of tumor necrosis factor α (TNFα) present in the supernatant of PBMCs after treatment with the bispecific antibodies described herein.

Mode for Carrying Out the Invention

[0021] Detailed Description of Embodiments The following description and examples illustrate in detail the embodiments of the present disclosure. It should be understood that the present disclosure is not limited to the specific embodiments described herein and may therefore vary. Those skilled in the art will recognize that numerous variations and modifications of the present disclosure exist and that they are encompassed within its scope.

[0022] All terms are intended to be understood as those skilled in the art would understand them. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which this disclosure pertains.

[0023] The headings of the sections used herein are for purposes of organization only and are not to be construed as limiting the subject matter described.

[0024] The various features of the present disclosure can be described in the context of a single embodiment, but they can also be provided separately or in any suitable combination. Conversely, the present disclosure can be described herein in the context of separate embodiments for clarity, but the present disclosure can also be implemented in a single embodiment. Definitions

[0025] The following definitions are supplementary to those skilled in the art and apply to this application, and should not be attributed to any related or unrelated cases, such as any generally owned patent or patent application. Any methods and materials similar to or equivalent to those described herein may be used in the practice for testing of this disclosure, but preferred materials and methods are described herein. Accordingly, the terms used herein are for the purpose of describing specific embodiments only and are not intended to be limiting.

[0026] In this application, the use of the singular form includes the plural form unless otherwise specifically stated. It should be noted that, as used herein, the singular forms "a," "an," and "the" refer to multiple objects unless otherwise explicitly stated in the text. In this application, the use of "or" means "and / or" unless otherwise stated. Furthermore, the use of the term "including" and other forms such as "include," "includes," and "included" is not limited.

[0027] Any reference herein to “some embodiments,” “a certain embodiment,” “one embodiment,” or “other embodiments” means that certain characteristics, structures, or features described in relation to that embodiment are included in at least some, but not all, embodiments of the Disclosure.

[0028] As used herein and in the claims, the words “comprising” (and any form of “comprise,” such as “comprise” and “comprises”), “having” (and any form of “have,” such as “have” and “has”), “including” (and any form of “includes,” such as “includes” and “include”), or “containing” (and any form of “contains,” such as “contain”) are comprehensive or open-ended and do not preclude any further unlisted elements or method steps. Any embodiment considered herein can be carried out with respect to any method or composition of the Disclosure, and vice versa. Furthermore, the methods of the Disclosure can be achieved using the compositions of the Disclosure.

[0029] The terms “about” or “approximately” mean within an acceptable margin of error for a particular value as determined by those skilled in the art, and will depend in part on how that value is measured or determined, i.e., on the limits of the measurement system. For example, “about” may mean within one or more standard deviations per practice in the art. Alternatively, “about” may mean a range of up to 20%, 10%, 5%, or 1% of a given value. In another example, the quantity “about 10” includes 10 and any quantities from 9 to 11. In yet another example, the term “about” in relation to a reference number may include values ​​within a range of plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of that value. Alternatively, particularly with respect to biological systems or processes, the term “about” may mean within one order of magnitude of a given value, preferably within five times, more preferably within two times. Where a specific value is stated in this application and claims, unless otherwise stated, the term “approximately” should be inferred to mean within the range of acceptable error for that specific value.

[0030] Where used herein, the terms “disease,” “disorder,” and “condition,” as used interchangeably herein, mean any alteration of the condition of any part of the body or organ, interruption or impairment of the performance of a function, and / or causing symptoms such as discomfort, dysfunction, pain, or even death to the person affected or to a person in contact with the person affected. Disease or disorder may also be associated with distemper, ailing, ailment, malady, disability, sickness, illness, complaint, or affectation.

[0031] As used herein, the term “needs it” means, when used in the context of therapeutic or preventive measures, for example, that a person at risk of developing the disease needs to have the disease, be diagnosed with the disease, or have the disease prevented. Thus, the subject who needs it may also be a subject who needs to have the disease treated or prevented.

[0032] As used herein, the term “administer” refers to the placement of a compound (e.g., an antibody or its antigen-binding fragment disclosed herein) by a method or route that results in at least partial delivery of the drug to a desired site. Pharmaceutical compositions comprising an antibody or its antigen-binding fragment disclosed herein may be administered by any suitable route that results in an effective treatment at the target, including, but not limited to, intravenous, intra-arterial, subcutaneous injection, or direct injection into tissue parenchyma. Where necessary or desired, administration may include, for example, intraventricular ("ICV") administration, intranasal administration, intracranial administration, intracavitary administration, intracerebellar administration, subcutaneous administration, or intrathecal administration.

[0033] As used herein, the terms “subject,” “patient,” “individual,” and similar terms are interchangeable and refer to vertebrates, mammals, primates, or humans. Mammals include, but are not limited to, humans, primates, rodents, wild or feral animals, agricultural animals, sport animals, and domesticated animals, including pets. Primates include, for example, chimpanzees, crab-eating macaques, spider monkeys, and macaques, such as rhesus macaques. Rodents include, for example, mice, rats, woodchucks, ferrets, rabbits, and hamsters. Domesticated and game animals include, for example, cattle, horses, pigs, deer, bison, buffalo, feline breeds, such as domestic cats, and canine breeds, such as dogs, foxes, and wolves, birds, such as chickens, emus, and ostriches, and fish, such as trout, catfish, and salmon. The terms “individual,” “patient,” and “subject” are interchangeable as used herein. The subject may be male or female. In some embodiments, the subject is a mammal. The mammal may be, but is not limited to, a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cattle. Non-human mammals may be advantageously used as subjects that are animal models of the condition or disorder. A non-limiting example is a mouse model. Furthermore, livestock and / or pets may be treated using the compositions and methods described herein. The subject may be a person who has been diagnosed with a given disorder, is currently being treated, or is seeking treatment, monitoring, adjustment or modification of an existing therapeutic treatment, or is at risk of developing the disorder.

[0034] As used herein, the terms “protein,” “peptide,” and “polypeptide” are used interchangeably and refer to a set of amino acid residues linked to one another by peptide bonds between the alpha-amino and carboxyl groups of adjacent residues. The terms “protein,” “peptide,” and “polypeptide” refer to polymers of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of their size or function. While “protein” and “polypeptide” are often used to refer to relatively large polypeptides, the term “peptide” is often used to refer to smaller polypeptides; however, there is overlap in the use of these terms in the art. The terms “protein,” “peptide,” and “polypeptide” are used interchangeably herein when referring to gene products and their fragments. These terms include, for example, natural and artificial proteins, protein fragments and polypeptide analogs of protein sequences (mutant proteins, variants, and fusion proteins, etc.), and proteins modified post-translationally or otherwise covalently or non-covalently. Peptides, polypeptides, or proteins may be monomers or polymers. Polypeptides may have amino acid sequences of naturally occurring polypeptides derived from any mammal. Such naturally occurring polypeptides can be isolated from nature or produced by recombinant or synthetic means. In some embodiments, the polypeptide is a “variant.” A “variant” means a biologically active polypeptide that, after aligning the sequence and introducing gaps as necessary to achieve maximum percent sequence identity, has at least about 80% amino acid sequence identity with the naturally occurring polypeptide, without considering any conservative substitutions as part of the sequence identity. Such variants include, for example, polypeptides that have one or more amino acid residues added or deleted at the N or C terminus of the polypeptide. In some embodiments, the variant has at least about 80% amino acid sequence identity. In some embodiments, the variant has at least about 90% amino acid sequence identity.In some embodiments, the variant has at least about 95% amino acid sequence identity with the native polypeptide. A "derivative" of a polypeptide is a polypeptide (e.g., an antibody) that has been chemically modified by conjugation, phosphorylation, and glycosylation to another chemical moiety (e.g., polyethylene glycol or albumin, e.g., human serum albumin).

[0035] As used herein, the term “percent identity” in the context of two or more nucleic acid or polypeptide sequences means two or more sequences or subsequences that have a specific percentage of identical nucleotide or amino acid residues when compared and aligned for maximum match, using one of the sequence comparison algorithms described below (e.g., using publicly available computer software such as BLAST, BLASTP, BLASTN, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, CLUSTAL OMEGA, or MUSCLE software, or other algorithms available to those skilled in the art), or measured by visual inspection. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (ncbi.nlm.nih.gov). Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required to achieve maximum alignment over the entire length of the sequences being compared. Depending on the application, the percentage of "identity" may exist across regions of the sequences being compared, for example, across functional domains, or across the entire length of the two sequences being compared. For sequence comparison, typically one sequence acts as the reference sequence compared to the test sequence. When using a sequence comparison algorithm, the test and reference sequences are entered into a computer, sub-sequence coordinates are specified as needed, and sequence algorithm program parameters are specified. The sequence comparison algorithm then calculates the percentage of sequence identity of the test sequence compared to the reference sequence based on the specified program parameters.Optimal alignment of sequences for comparison can be performed, for example, by the partial homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), the similarity search method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see Ausubel et al., cited above).

[0036] As used herein, the terms “increased,” “increased,” and “enhance” mean an increase by a statistically significant amount; to avoid any doubt, the terms “increased,” “increased,” or “enhance” mean an increase of at least 10% compared to a reference level, for example, an increase of at least about 10%, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or 100% compared to a reference level, or any increase between 10% and 100%, or an increase of at least about 2 times, or at least about 3 times, or at least about 4 times, or at least about 5 times, or at least about 10 times, or any increase between 2 times and 10 times, or more.

[0037] As used herein, the term “antibody” refers, in the present examples, to an immunoglobulin molecule that specifically binds to or immunologically reacts to a particular antigen, which may be, for example, TREM1, an interleukin of the IL-17 family, or IL-17R. Antibodies may include, for example, polyclonal antibodies, monoclonal antibodies, genetically engineered antibodies, and their antigen-binding fragments. Antibodies may be, for example, mouse, chimeric, humanized, heteroconjugate, bispecific, diabody, triabody, or tetrabody. Antigen-binding fragments may include, for example, Fab', F(ab')2, Fab, Fv, rIgG, scFv, hcAb (heavy chain antibody), single-domain antibody, V HH , V NAR The antibody may contain sdAb or nanobodies. The term “monoclonal antibody,” as used herein, refers to an antibody produced by a single clone of a B cell and bound to the same epitope. In contrast, “polyclonal antibody” refers to a group of antibodies produced by different B cells and bound to different epitopes of the same antigen. The total antibody may contain four polypeptides: two identical copies of a heavy (H) chain polypeptide and two identical copies of a light (L) chain polypeptide. Each heavy chain may contain one N-terminal variable (VH) region and three C-terminal constant (CH1, CH2, and CH3) regions, and each light chain may contain one N-terminal variable (VL) region and one C-terminal constant (CL) region. The variable regions of each pair of light and heavy chains may form the antigen-binding site of the antibody. In exemplary embodiments of bispecific antibodies, multiple different antigen-binding sites may be present. The VH and VL regions may have a similar general structure, with each region containing four relatively conserved framework regions. In some embodiments, the framework region may be connected by three complementarity-determining regions (CDRs). In some embodiments, the three CDRs, known as CDR1, CDR2, and CDR3, form a “hypervariable region” of the antibody responsible for antigen binding.

[0038] As used herein, the term “chimeric antibody” refers to an antibody in which a portion of the heavy chain and / or light chain originates from a particular source or species, but the remaining portion of the heavy chain and / or light chain originates from a different source or species.

[0039] As used herein, the term “human antibody” means an antibody that contains the amino acid sequence corresponding to that of an antibody produced by a human or human cell, or derived from a non-human source that utilizes the human antibody repertoire or a human antibody coding sequence (e.g., obtained from a human source or designed de novo).

[0040] As used herein, the term “humanized antibody” refers to an amino acid sequence that differs from that of an antibody derived from a non-human species by one or more amino acid substitutions, deletions, and / or additions such that, when administered to a human subject, it is less likely to induce an immune response and / or induces a less vigorous immune response compared to an antibody of a non-human species. In some embodiments, certain amino acids in the heavy and / or light chain framework and constant domain of a non-human species antibody are mutated to produce a humanized antibody. In some embodiments, a constant domain derived from a human antibody is fused to a variable domain of a non-human species. In other embodiments, one or more amino acid residues in one or more CDR sequences of a non-human antibody are altered to reduce the possible immunogenicity of the non-human antibody when administered to a human subject, and the altered amino acid residues are not important for the antibody’s immune-specific binding to its antigen, or the alteration to the resulting amino acid sequence is a conservative alteration, and as a result, the binding of the humanized antibody to the antigen is not significantly worse than the binding of the non-human antibody to the antigen. Examples of methods for producing humanized antibodies can be found in U.S. Patents 6,054,297, 5,886,152, and 5,877,293. For further details, see Jones et al., Nature, 1986, 321:522-525; Riechmann et al., Nature, 1988, 332:323-329; and Presta, Curr. Op. Struct. Biol., 1992, 2:593-596, which are incorporated in their entirety by reference.

[0041] As used herein, the term “epitope” refers to a portion of an antigen that specifically binds to an antibody. Epitopes often consist of surface-accessible amino acid residues and / or sugar side chains and may have specific three-dimensional structural properties as well as specific electrostatic properties. Constructive and non-constructive epitopes are distinguished in that binding to the former, rather than the latter, may be lost in the presence of a denaturing solvent. Epitopes may include amino acid residues directly involved in binding and other amino acid residues not directly involved in binding. The epitope to which an antibody binds can be determined using known techniques for epitope determination, such as tests for antibody binding to TREM1 or its variants, IL-17 or its variants, IL-17R or its variants, or combinations thereof.

[0042] As used herein, the term “complementarity-determining regions” (CDRs, i.e., CDR1, CDR2, and CDR3) refers to amino acid residues in an antibody variable domain whose presence is required for antigen binding. Each variable domain typically has three CDR regions, identified as CDR1, CDR2, and CDR3. The CDRs of the variable heavy chain may be CDR-H1, CDR-H2, and CDR-H3. The CDRs of the variable light chain may be CDR-L1, CDR-L2, and CDR-L3. Exemplary hypervariable loops are located at amino acid residues 26–32 (L1), 50–52 (L2), 91–96 (L3), 26–32 (H1), 53–55 (H2), and 96–101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) are located at amino acid residues 24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 of H2, and 95-102 of H3 (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. (1991)). Thus, HV may be contained within the corresponding CDR, and the references herein to the "hypervariable loops" of the VH and VL domains should be interpreted as also encompassing the corresponding CDRs, and vice versa, unless otherwise noted. The more highly conserved regions of the variable domains are called framework regions (FRs), as defined below. The variable domains of the natural heavy and light chains each contain four FRs (FR1, FR2, FR3, and FR4, respectively) and primarily adopt a [beta]sheet structure connected by three hypervariable loops. The hypervariable loops in each chain are held together in close proximity by the FRs, while the hypervariable loops originating from other chains contribute to the formation of the antibody's antigen-binding site.Structural analysis of antibodies has demonstrated the relationship between the sequence and shape of binding sites formed by complementarity-determining regions (Chothia et al., J. Mol. Biol. 227: 799-817 (1992)); Tramontano et al., J. Mol. Biol, 215: 175-182 (1990)). Despite their high sequence variability, five of the six loops adopt only a small repertoire of backchain conformations, known as the "standard structure." These conformations are determined first by the length of the loops, second by the presence of key residues at specific positions within the loops and framework regions, and by their ability to pack, hydrogen bond, or adopt atypical backchain conformations. The antibodies or antigen-binding fragments of the present disclosure may include a CDR3 region having a length of at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. The antibodies or antigen-binding fragments of the present disclosure may also include a CDR3 region having a length of at least about 18 amino acids.

[0043] As used herein, the term “variable region” used in reference to an antibody refers, alone or in combination, to the variable region of the antibody light chain or the variable region of the antibody heavy chain. Each of the heavy and light chain variable regions consists of four framework regions (FRs) connected by three complementarity-determining regions (CDRs), also known as hypervariable regions. The CDRs in each chain are held together in close proximity by the FRs, and CDRs from other chains contribute to the formation of the antibody’s antigen-binding site. At least two techniques exist for determining CDRs: (1) a method based on interspecific sequence variability (i.e., Kabat et al. Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda Md.)); and (2) a method based on crystallographic studies of antigen-antibody complexes (Allazikani et al (1997) J. Molec. Biol. 273:927-948). CDRs may refer to those defined by either of these methods or a combination of both. The six hypervariable loops (three of which originate from the heavy and light chains, respectively) contribute amino acid residues for antigen binding, giving the antibody antigen-binding specificity. However, even a single variable domain (or half of the Fv containing only the three antigen-specific CDRs) has the ability to recognize and bind to the antigen, though with lower affinity than the entire binding site.

[0044] As used herein, the term “constant region” used in reference to an antibody refers, alone or in combination, to the constant region of the antibody light chain (i.e., the light chain constant region) or the constant region of the antibody heavy chain (i.e., the heavy chain constant region). The constant region does not change with respect to antigen specificity.

[0045] As used herein, the term “heavy chain region” includes an amino acid sequence derived from the constant domain of an immunoglobulin heavy chain. A polypeptide comprising a heavy chain region includes at least one of the following: the CH1 domain, the hinge (e.g., upper, central, and / or lower hinge regions) domain, the CH2 domain, the CH3 domain, or a variant or fragment thereof. In some embodiments, the antibody or its antigen-binding fragment may include the Fc region of the immunoglobulin heavy chain (e.g., the hinge portion, the CH2 domain, and the CH3 domain). In other embodiments, the antibody or its antigen-binding fragment lacks at least one region of the constant domain (e.g., all or part of the CH2 domain). In some embodiments, at least one, preferably all, of the constant domains is derived from a human immunoglobulin heavy chain. For example, in one preferred embodiment, the heavy chain region includes a full human hinge domain. In other preferred embodiments, the heavy chain region includes a full human Fc region (e.g., the hinge, CH2, and CH3 domain sequences derived from human immunoglobulin). In some embodiments, the constituent constant domains of the heavy chain region are derived from different immunoglobulin molecules.

[0046] As used herein, the term “hinge region” includes a region of the heavy chain molecule that binds the CH1 domain to the CH2 domain. The hinge region may contain approximately 25 residues, is flexible, and thus allows the two N-terminal antigen-binding regions to move independently. The hinge region can be subdivided into three distinct domains: upper, central, and lower hinge domains (Roux et al. J. Immunol. 1998 161:4083).

[0047] As used herein, the term "Fv" refers to the smallest antibody fragment containing a complete antigen recognition and binding site. This fragment consists of a dimer of one heavy chain and one light chain variable region domain in a tight, non-covalent association. From the folding of these two domains, six hypervariable loops (three derived from the H and L chains, respectively) are generated, contributing amino acid residues for antigen binding and conferring antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific CDRs) has the ability to recognize and bind to the antigen, though with lower affinity than the entire binding site.

[0048] As used herein, the term “heavy chain variable region” or “VH” refers, when used in reference to an antibody, to a heavy chain fragment containing three CDRs positioned between flanking stretches known as framework regions, where these framework regions are generally more conserved than the CDRs and form a scaffold for supporting the CDRs.

[0049] As used herein, the term “light chain variable region” or “VL” refers, when used in reference to an antibody, to a fragment of a light chain containing three CDRs positioned between flanking stretches, known as a framework region, where these framework regions are generally more conserved than the CDRs and form a scaffold for supporting the CDRs.

[0050] As used herein, the term "framework residue" or "FR" refers to a variable domain amino acid residue other than a hypervariable region amino acid residue.

[0051] As used herein, the term “antibody heavy chain” refers to the larger of the two polypeptide chains present in an antibody molecule in its naturally occurring conformation, and typically determines the class to which the antibody belongs.

[0052] As used herein, the term “antibody light chain” refers to the smaller of the two polypeptide chains present in an antibody molecule in its naturally occurring conformation. Kappa ("κ") and lambda ("λ") light chains refer to the two main antibody light chain isotypes.

[0053] As used herein, the terms “specifically bind” or “preferentially bind” mean that an antibody or its antigen-binding fragment binds to a target with higher affinity and / or avidity than it would to bind to an epitope on an unrelated polypeptide. The specificity of an antibody or its antigen-binding fragment or portion can be determined based on affinity and / or avidity. Methods for determining such specific binding are also well known in the art.

[0054] As used herein, the term “multispecific antibody” is an antibody comprising two or more different antigen-binding domains that collectively and specifically bind to two or more different epitopes. These two or more different epitopes may be epitopes on the same cell or on different cells. In some embodiments, the multispecific antibody binds to two different epitopes (i.e., a “bispecific antibody”). In some embodiments, the multispecific antibody binds to three different epitopes (i.e., a “trispecific antibody”).

[0055] As used herein, “recombinant antibody” is an antibody comprising an amino acid sequence derived from two different species or two different sources, and comprising a molecule that is synthetic and / or not naturally occurring. In non-limiting examples, recombinant antibodies may include antibodies having a non-human CDR and human variable region framework or constant or Fc region, an antibody having a binding domain derived from two different monoclonal antibodies, or an antibody comprising mutations in one or more amino acid residues that increase or decrease the biological activity or binding of a portion of the antibody. In certain embodiments, recombinant antibodies are produced from or synthesized from recombinant DNA molecules. In certain embodiments, the antibodies described herein are polypeptides encoded by one or more polynucleotides.

[0056] As used herein, “recognize,” “bind,” or “selective” refers to the association or binding between an antigen-binding domain and an antigen. As used herein, “antigen” refers to an antigenic substance that can elicit an immune response in a host. The antigenic substance may be a molecule, such as a co-stimulatory molecule, that can elicit an immune response in a host.

[0057] As used herein, “antibody construct” means a construct which may contain an antigen-binding domain and an Fc domain.

[0058] As used herein, “binding domain” refers to an antibody or non-antibody domain.

[0059] As used herein, “antigen-binding domain” refers to a binding domain derived from an antibody or from a non-antibody capable of binding to an antigen. If there are more than one antigen-binding domains in a given conjugate or antibody construct, the antigen-binding domains may be numbered (e.g., the first antigen-binding domain, the second antigen-binding domain, the third antigen-binding domain, etc.). Different antigen-binding domains in the same conjugate or construct may target the same or different antigens.

[0060] As used herein, "antibody-antigen binding domain" refers to a binding domain derived from an antibody capable of binding to an antigen.

[0061] As used herein, “Fc domain” refers to an Fc domain derived from an antibody or non-antibody capable of binding to an Fc receptor. As used herein, “Fc domain” and “Fc-containing domain” are interchangeable.

[0062] As used herein, “target-binding domain” refers to a construct containing an antigen-binding domain derived from an antibody or from a non-antibody capable of binding to an antigen.

[0063] As used herein, the omissions for natural 1-enantiomer amino acids remain the same and may be as follows: alanine (A, Ala); arginine (R, Arg); asparagine (N, Asn); aspartic acid (D, Asp); cysteine ​​(C, Cys); glutamic acid (E, Glu); glutamine (Q, Gln(Gin)); glycine (G, Gly); histidine (H, His); isoleucine (I, Ile(He)); leucine (L, Leu); lysine (K, Lys); methionine (M, Met); phenylalanine (F, Phe); proline (P, Pro); serine (S, Ser); threonine (T, Thr); tryptophan (W, Trp); tyrosine (Y, Tyr); valine (V, Val). Unless otherwise specified, X may represent any amino acid.

[0064] The term "pharmaceutically acceptable" is used herein to mean a compound, material, composition, and / or dosage form that, within the bounds of sound medical judgment, is suitable for use in contact with subjects, such as human and animal tissues, without exhibiting excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit-to-risk ratio.

[0065] As used herein, the terms “pharmaceutically acceptable excipient” or “pharmaceutically acceptable carrier” mean a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Each carrier must be “acceptable” in the sense that it is compatible with the other components of the formulation and must not be harmful to the patient. Some examples of materials that may function as pharmaceutically acceptable carriers include: (1) sugars, e.g., lactose, glucose and sucrose; (2) starches, e.g., corn starch and potato starch; (3) cellulose and its derivatives, e.g., sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; (4) tragacanth powder; (5) malt; (6) gelatin; (7) talc; (8) excipients, e.g., cocoa butter and suppository wax; (9) oils, e.g., peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil. Examples include oils; (10) glycols, e.g., propylene glycol; (11) polyols, e.g., glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters, e.g., ethyl oleate and ethyl laurate; (13) agar; (14) buffers, e.g., magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) water free of pyrogens; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solution; and (21) other non-toxic suitable substances used in pharmaceutical formulations.

[0066] Antigens can elicit an immune response. Antigens may be proteins, polysaccharides, lipids, or glycolipids that can be recognized by immune cells such as T cells or B cells. Exposure of immune cells to one or more of these antigens can trigger a rapid cell division and differentiation response, potentially leading to the formation of clones of the exposed T cells and B cells. B cells can differentiate into plasma cells, which can then produce antibodies that selectively bind to the antigen.

[0067] The term "antigen recognition portion" or "antibody recognition domain" refers to a molecule or part of a molecule that specifically binds to an antigen. In one embodiment, the antigen recognition portion is an antibody, an antibody-like molecule, or a fragment thereof, and the antigen is an exogenous antigen or an infectious disease antigen.

[0068] The terms “antibody fragment,” “antibody fragment,” “functional fragment of an antibody,” “antigen-binding domain,” or their grammatical equivalents are used interchangeably herein and mean one or more fragments or portions of an antibody that retain the ability to specifically bind to an antigen (see Holliger et al., Nat. Biotech., 23(9):1126-1129 (2005) for general reference). An antibody fragment preferably comprises, for example, one or more CDRs, variable regions (or parts thereof), constant regions (or parts thereof), or a combination thereof. Examples of antibody fragments include, but are not limited to, (i) Fab fragments, which are monovalent fragments that may contain VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, which are bivalent fragments containing two Fab fragments linked by disulfide crosslinks in the stem region; (iii) Fv fragments, which consist of the VL and VH domains of a single arm of the antibody; and (iv) single-chain Fv(scFv), which are monovalent molecules consisting of two domains of an Fv fragment (i.e., VL and VH) linked by a synthetic linker that allows the two domains to be synthesized as a single polypeptide chain (e.g., Bird et al., Science, 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA, 85: 5879-5883 (1988); and Osbourn et al., Nat. Biotechnol., 16: 778). (See 1998) and (v) each polypeptide chain may include a VH connected to VL by a peptide linker that is too short to allow pairing between VH and VL on the same polypeptide chain, thereby inducing pairing between complementary domains on different VH-VL polypeptide chains to produce a dimeric molecule having two functional antigen-binding sites. Antibody fragments are known in the art and are described in detail, for example, by U.S. Patent No. 8,603,950. Other antibody fragments may include variable fragments (VHH) of heavy chain antibodies.

[0069] As used herein, the term "Fab" refers to a region of an antibody (monovalent antigen-binding fragment) consisting of one constant domain and one variable domain in both the heavy and light chains, wherein the heavy chain is truncated to lack the CH2 and CH3 domains (i.e., VH, CH1, VL, and CL), and may also lack some or all of the hinge region. It can be produced by digestion of the whole antibody with papain enzyme. Fab may refer to this region separately, or to this region in the context of a full-length antibody, immunoglobulin construct, or Fab fusion protein. Fab can be obtained by treating the whole antibody with pepsin and then reducing it to obtain a molecule consisting of the intact light chain and a portion of the heavy chain containing VH and a single constant domain. Two Fab' fragments are obtained per antibody treated in this manner.

[0070] As used herein, the term “scFv” refers to an antibody fragment containing the VH and VL domains of an antibody, where these domains are present within a single polypeptide chain. See, for example, U.S. Patents 4,946,778, 5,260,203, 5,455,030, and 5,856,456. Generally, Fv polypeptides further contain a polypeptide linker between the VH and VL domains, enabling the scFv to form a desirable structure for antigen binding. For an overview of scFv, see Pluckthun (1994) The Pharmacology of Monoclonal Antibodies vol 113 ed. Rosenburg and Moore (Springer-Verlag, New York) pp 269-315. The VH and VL domain complex of the Fv fragment can also be stabilized by a disulfide bond (U.S. Patent 5,747,654).

[0071] The term "conservative amino acid substitution" or "conservative mutation" refers to the substitution of one amino acid with another amino acid that shares common properties. A functional method for defining the common properties between individual amino acids is to analyze the normalized frequency of amino acid changes between corresponding proteins in the same species (Schulz, GE and Schirmer, RH, Principles of Protein Structure, Springer-Verlag, New York (1979)). According to such analysis, a group of amino acids can be defined if the amino acids within the group preferentially exchange with each other and therefore many are similar in their impact on the overall protein structure. Examples of conservative mutations include amino acid substitutions of amino acids within the above subgroups, e.g., the substitution of lysine with arginine and vice versa, which can maintain a positive charge; the substitution of glutamate with aspartate and vice versa, which can maintain a negative charge; the substitution of serine with threonine, which can maintain a free -OH group; and the substitution of glutamine with asparagine, which can maintain a free -NH2 group. Alternatively, or furthermore, the therapeutic agent may include the amino acid sequence of a reference protein having at least one non-conservative amino acid substitution.

[0072] The terms "non-conservative mutation" or "non-conservative amino acid substitution" include amino acid substitutions between different groups, such as the substitution of lysine for tryptophan or phenylalanine for serine. In this case, it is preferable that the non-conservative amino acid substitution does not interfere with or inhibit the biological activity of the therapeutic agent. The non-conservative amino acid substitution may enhance the biological activity of the therapeutic agent so that its biological activity is increased compared to the wild-type therapeutic agent.

[0073] A "multispecific antibody" is an antibody that can simultaneously bind to at least two targets of different structures, such as two different antigens, two different epitopes on the same antigen, or haptens and / or antigens or epitopes. A "multivalent antibody" is an antibody that can simultaneously bind to at least two targets of the same or different structures. Valency indicates how many binding arms or sites an antibody has for a single antigen or epitope; i.e., whether it is monovalent, bivalent, trivalent, or polyvalent. The polyvalent nature of an antibody means that it can utilize multiple interactions in binding to an antigen, thus increasing its avidity of binding to the antigen. Specificity indicates how many antigens or epitopes an antibody can bind to, i.e., whether it is monospecific, bispecific, trispecific, or multispecific. Using these definitions, a natural antibody is bivalent because it has two binding arms, but monospecific because it binds to one epitope. A multispecific, multivalent antibody is a construct having more than one binding region of different specificities. For example, the bispecific antibody construct disclosed herein has a first antigen-binding region and a second antigen-binding region, where the first and second antigen-binding regions are different.

[0074] A "bispecific antibody" is an antibody that can simultaneously bind to two targets having different structures. A bispecific antibody (bsAb) and a bispecific antibody fragment (bsFab) may have, for example, at least one arm (or binding domain) that specifically binds to a first antigen, and at least one other arm (or binding domain) that specifically binds to a second antigen. At least one of the first and second antigens may be an antigen produced by or associated with diseased cells, tissues, organs, or pathogens. A variety of bispecific antibodies can be produced using molecular engineering.

[0075] As used herein, the term “vector” refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is ligated. This term includes vectors as self-replicating nucleic acid structures, as well as vectors that are incorporated into the genome of a host cell into which they are introduced. Certain vectors can direct the expression of the nucleic acid to which they are operably ligated. Such vectors are referred herein to as “expression vectors.”

[0076] As used herein, the terms “host cell,” “host cell line,” and “host cell culture” are interchangeable and refer to cells into which exogenous nucleic acids have been introduced, and the offspring of such cells. Host cells include “transformed” (or “transformed cell”) and “transfectant” (or “transfected cell”), respectively, including primary transformed or transfected cells and their offspring. Such offspring may not be exactly identical to the parent cells in nucleic acid content and may contain mutations.

[0077] A bispecific antibody construct, or any composition described herein, is said to be administered in a “therapeutic effective dose” if the amount administered is physiologically significant. A drug is physiologically significant if its presence results in a detectable physiological change in the recipient. In certain embodiments, a bispecific antibody construct disclosed herein is physiologically significant if its presence induces a response or alleviates signs and symptoms of an infectious or autoimmune disease condition. A physiologically significant effect may also be the induction of a humoral and / or cellular immune response in the recipient.

[0078] The term “linker” is used to describe a polypeptide containing two or more amino acid residues linked by a peptide bond, used to link one or more antigen-binding moieties or variable domains. Such linker polypeptides are well known in the art (see, for example, Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, RJ, et al. (1994) Structure 2:1121-1123). In some embodiments, the linker peptide contains an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 44. In some embodiments, the linker peptide includes an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 45.

[0079] An "Fv" or "Fv fragment" may consist only of the light chain variable domain (VL) and heavy chain variable domain (VH) of a "single arm" of immunoglobulin. Thus, an "Fv" is the smallest antibody fragment containing a complete antigen recognition site and binding site. A "two-chain" Fv fragment consists of a dimer of one heavy chain and one light chain variable domain in a tight, non-covalent association. A single-chain Fv species (scFv) may contain the VH and VL domains of immunoglobulin, which are located in a single polypeptide chain that is covalently linked to each other by a linker peptide. Typically, in an scFv fragment, the light and heavy chain variable domains associate in a dimer structure similar to that of a two-chain Fv species. A single-chain Fv fragment may have either a variable light chain domain located at the N-terminus of a single polypeptide chain, followed by a variable heavy chain domain located at the C-terminus of a linker and polypeptide chain, or vice versa, with a linker peptide positioned between the N-terminus and C-terminus. The linker peptide may be any flexible linker known in the art, for example, made from glycine and serine residues. The domain association between the VH and VL domains can also be further stabilized by introducing disulfide bonds into the conserved framework region (see Reiter et al. Stabilization of the Fv fragments in recombinant immunotoxins by disulfide bonds engineered into conserved framework regions, Biochemistry 1994, 33, 6551-5459). Such scFv fragments are also known as disulfide-stabilized scFv fragments (ds-scFv).

[0080] As used herein, the term “to treat” (and its variations such as “to treat” or “treatment”) refers to a clinical intervention in an attempt to alter the natural course of a disease or condition in a subject requiring it. Treatment may be performed during the course of clinicopathology. Desired effects of treatment include the cure (if applicable), delay of onset, reduction, alleviation, or improvement of one or more symptoms of the disease, improvement of the disease, or reduction or improvement of any associated symptoms of the disease or predisposition to the onset of the disease.

[0081] As used herein, the term “sufficient amount” means an amount sufficient to produce the desired effect, for example, an amount sufficient to modulate the immune response in the subject.

[0082] As used herein, the terms “modulate” and “modulation” refer to the reduction or inhibition, or the activation or increase, of the variable described. functional antibody fragment

[0083] Functional antibody fragments bind to target proteins. In some embodiments, functional antibody fragments promote the degradation of target proteins. In some embodiments, functional antibody fragments induce the degradation of target proteins. In some embodiments, functional antibody fragments induce / promote the cleavage of target proteins. In some embodiments, functional antibody fragments induce / promote the internalization of target proteins. In some embodiments, functional antibody fragments induce / promote the shedding of target proteins. In some embodiments, functional antibody fragments induce / promote the downregulation of target protein expression. In some embodiments, functional antibody fragments prevent the target protein-mediated activity of one or more downstream signaling proteins. In some embodiments, functional antibody fragments prevent the target protein-mediated expression of one or more downstream signaling proteins. In some embodiments, the target proteins are directly and / or indirectly associated with inflammation. Therefore, in some embodiments, administration of a functional antibody fragment in a subject results in reduced inflammation compared to inflammation in the subject before administration of the functional antibody fragment. In some embodiments, the target protein includes TREM1, a cytokine from the IL-17 family, IL-17R, or a combination thereof.

[0084] Functional antibody fragments that recognize specific epitopes can be generated by known techniques. Functional antibody fragments are, for example, the antigen-binding portions of antibodies such as F(ab')2, Fab', F(ab)2, Fab, Fv, and scFv. The F(ab')2 fragment can be produced by pepsin digestion of the antibody molecule, and the Fab' fragment can be generated by reducing the disulfide crosslinks of the F(ab')2 fragment. Alternatively, a Fab' expression library can be constructed (Huse et al., 1989, Science, 246:1274-1281) to enable rapid and easy identification of monoclonal Fab' fragments with desired specificity. The F(ab)2 fragment can be generated by papain digestion of the antibody.

[0085] Single-chain Fv molecules (scFv) may contain a VL domain and a VH domain. The VL and VH domains associate to form a target binding site. These two domains can be further covalently bonded by a peptide linker (L). Methods for constructing scFv molecules and designing suitable peptide linkers are described in U.S. Patent No. 4,704,692; U.S. Patent No. 4,946,778; Raag and Whitlow, FASEB 9:73-80 (1995); and Bird and Walker, TIBTECH, 9:132-137 (1991).

[0086] Furthermore, techniques for producing single-domain antibodies (DABs or VHHs) are known in the art, for example, as disclosed in Cossins et al. (2006, Prot Express Purif 51:253-259), which is incorporated herein by reference. Single-domain antibodies can be obtained from camels, alpacas, or llamas, for example, by standard immunization techniques (see, e.g., Muyldermans et al., TIBS 26:230-235, 2001; Yau et al., J Immunol Methods 281:161-75, 2003; Maass et al., J Immunol Methods 324:13-25, 2007). VHHs may have potent antigen-binding capabilities and can interact with novel epitopes that are inaccessible to conventional VH-VL pairs (Muyldermans et al., 2001). Alpaca serum IgG contains approximately 50% IgG antibodies (HCAb) consisting solely of camelid heavy chain IgG (Maass et al., 2007). By immunizing alpacas with known antigens such as TNF-α, VHH that binds to and neutralizes the target antigen can be isolated (Maass et al., 2007). PCR primers that amplify substantially all alpaca VHH coding sequences have been identified, and these can be used to construct an alpaca VHH phage display library that can be used for isolation of antibody fragments by standard biopanning techniques well known in the industry (Maass et al., 2007). In certain embodiments, VHH antibody fragments can be used in the claimed compositions and methods.

[0087] Antibody fragments can be prepared by proteolytic hydrolysis of full-length antibodies or by expression of DNA encoding antibody fragments in E. coli or another host. Antibody fragments can also be obtained by pepsin or papain digestion of full-length antibodies by conventional methods. These methods are described, for example, by Goldenberg's U.S. Patents No. 4,036,945 and 4,331,647 and the references contained therein. See also Nisonoff et al., Arch Biochem. Biophys. 89: 230 (1960); Porter, Biochem. J. 73: 119 (1959), Edelman et al., in METHODS IN ENZYMOLOGY VOL. 1, page 422 (Academic Press 1967), and Coligan at pages 2.8.1-2.8.10 and 2.10-2.10.4. multispecific antibodies

[0088] A multispecific antibody is an antibody that can bind to at least two different targets. In some embodiments, the at least two different targets include two different epitopes. In some embodiments, the two different epitopes are TREM1 or a variant thereof, and IL-17 or a variant thereof or its receptor.

[0089] Methods for producing multispecific antibodies are well known in the industry. Traditionally, recombinant production of multispecific antibodies is based on the simultaneous expression of two immunoglobulin heavy / light chain pairs, each having two heavy chains with different specificities (Milstein and Cuello, Nature, 305:537-539 (1983)). Precise molecular purification is usually achieved by affinity chromatography. Similar procedures are disclosed in WO93 / 08829 and Traunecker et al., EMBO J., 10:3655-3659 (1991).

[0090] There are five major antibody classes: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. Therefore, the multispecific antibodies described herein include the kappa constant region, lambda constant region, alpha constant region, gamma constant region, delta constant region, epsilon constant region, mu constant region, their functional fragments, or combinations thereof.

[0091] The class of an antibody or immunoglobulin refers to the type of constant domain or region in its heavy chain. In some embodiments, the heavy chain is IgA. In some embodiments, the heavy chain is IgD. In some embodiments, the heavy chain is IgE. In some embodiments, the heavy chain is IgG. In some embodiments, the heavy chain is IgM. In some embodiments, the heavy chain is IgG1. In some embodiments, the heavy chain is IgG2. In some embodiments, the heavy chain is IgG3. In some embodiments, the heavy chain is IgG4. In some embodiments, the heavy chain is IgA1. In some embodiments, the heavy chain is IgA2. In some embodiments, the antibody is an IgG1 antibody.

[0092] In some embodiments, the antibody is an IgG3 antibody. In some embodiments, the antibody is an IgG2 antibody. In some embodiments, the antibody is an IgG4 antibody.

[0093] In some embodiments, the multispecific antibodies described herein include a light chain. In some embodiments, the light chain includes a kappa light chain or a lambda light chain. Multispecific antibodies, such as kappa or lambda antibodies, can be prepared using any of a variety of art-recognized techniques, including those disclosed in WO2012 / 023053, the entirety of which is incorporated herein by reference.

[0094] In some embodiments, a variable antibody domain having a desired binding specificity (antibody-antigen binding site) can be ligated to an immunoglobulin constant domain sequence to form a bispecific antibody. In some embodiments, the fusion is preferably with an immunoglobulin heavy chain constant domain including at least a portion of the hinge, CH2, and CH3 regions. In some embodiments, it is preferable to have a first heavy chain constant region (CH1) containing a site necessary for light chain binding present in at least one of the fusions. The immunoglobulin heavy chain fusions and, optionally, DNA encoding the immunoglobulin light chain can be inserted into separate expression vectors and co-transfected into a suitable host organism. For further details on the generation of bispecific antibodies, see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986), the contents of which are incorporated herein by reference.

[0095] In some embodiments, the interface between pairs of antibody molecules in the constructs herein is manipulated to maximize the percentage of heterodimers recovered from recombinant cell cultures. In this method, one or more smaller amino acid side chains originating from the interface of the first antibody molecule are replaced with larger side chains to form a protrusion or knob (e.g., tyrosine or tryptophan). By replacing the larger amino acid side chain with a smaller one (e.g., alanine or threonine), a compensatory cavity or hole of the same or similar size as the larger side chain is created on the interface of the second antibody molecule. This provides a mechanism for increasing the yield of heterodimers over other undesirable end products such as homodimers.

[0096] Techniques for generating bispecific antibodies from functional antibody fragments are described in the literature. For example, bispecific antibodies can be prepared using chemical bonding. The produced bispecific antibodies can be used as agents for the selective immobilization of enzymes.

[0097] Various techniques for directly producing and isolating functional bispecific antibody fragments from recombinant cell cultures are also described. For example, bispecific antibodies were produced using leucine zippers. Kostelny et al., J. Immunol. 148(5):1547-1553 (1992). Leucine zipper peptides derived from Fos and Jun proteins were ligated to the Fab' portion of two different antibodies by gene fusion. The antibody homodimer was reduced at the hinge region to form a monomer, which was then reoxidized to form an antibody heterodimer. This method can also be used for the production of antibody homodimers. The “diabody” technique described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) provided an alternative mechanism for producing functional bispecific antibody fragments. The functional fragment contains a heavy chain variable domain (VH) connected to a light chain variable domain (VL) by a linker that is too short to allow pairing between the two domains on the same chain. Thus, the VH and VL domains of one functional fragment are forced to pair with the complementary VL and VH domains of another functional fragment, thereby forming two antigen-binding sites. Another strategy for producing functionally bispecific antibody fragments using single-chain Fv(sFv) dimers has also been reported. See Gruber et al., J. Immunol. 152:5368 (1994).

[0098] Antibodies with a valency greater than 2 are intended. For example, triplicate antibodies can be prepared. Tutt et al., J. Immunol. 147:60 (1991). Exemplary bispecific antibodies can bind to two different epitopes, at least one of which is derived from the protein antigen of the present invention. Alternatively, an anti-antigenic arm of an immunoglobulin molecule can be mixed with an arm that binds to a catalyst on a leukocyte, such as a T cell receptor molecule (e.g., CD2, CD3, CD28, or B7), or an Fc receptor for IgG (FcγR), such as FcγRI(CD64), FcγRII(CD32), and FcγRIII(CD16), to concentrate the cellular defense mechanism on cells expressing a specific antigen. Bispecific antibodies can also be used to direct cytotoxic agents to cells expressing a specific antigen. These antibodies may have an antigen-binding arm and an arm that binds to a cytotoxic agent or radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA. The other bispecific antibody of interest binds to the protein antigen described herein and further binds to tissue factor (TF).

[0099] Such multispecific molecules (e.g., bispecific, triplicate molecules) have been generated using several strategies, including chemical crosslinking of functional antibody fragments, forced heterodimerization, quadroma technology, fusion of functional antibody fragments via polypeptide linkers, and the use of single-domain antibodies. The availability of recombinant DNA technology has led to the generation of multiple bispecific antibody forms (see, e.g., Ridgway JB et al. (1996) Protein Eng 9: 617-621). Linkers and mutations are often introduced into different regions of antibodies to force heterodimerization or to connect different binding sites within a single molecule. IL-17 and IL-17 family structures

[0100] The IL-17 family in humans includes IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, and IL-17F. These cytokines are involved in inflammatory responses and can mediate or induce the expression of various other cytokines, factors, and mediators, including tissue necrosis factor-alpha (TNF-α), IL-6, IL-8, IL-1β, granulocyte colony-stimulating factor (G-CSF), prostaglandin E2 (PGE2), IL-10, IL-12, IL-1R antagonists, leukemia inhibitors, and stromelicin (Yao et al., J. Immunol., 155(12): 5483-5486 (1995); Fossiez et al., J. Exp. Med., 183(6): 2593-2603 (1996); Jovanovic et al., J. Immunol., 160: 3513-3521 (1998); Teunissen et al., J. Investig. Dermatol., 111: 645-649 (1998); Chabaud et al., J. Immunol., 161: 409-414 (1998)). IL-17 also induces nitric oxide in chondrocytes and human osteoarthritis explants (Shalom-Barak et al., J. Biol. Chem., 273: 27467-27473 (1998); Attur et al., Arthritis Rheum., 40: 1050-1053 (1997)). The amino acid sequences of IL-17A, IL-17B, IL-17C, IL-17-D, IL-17E, and IL-17F are listed in Table 1. [Table 1-1] [Table 1-2]

[0101] IL-17 can induce the release of cytokines, chemokines, and growth factors, and is an important local orchestrator for neutrophil accumulation, playing a role in cartilage and bone destruction. IL-17 signaling has been thought to play a role in various autoimmune diseases, including rheumatoid arthritis (RA), ankylosing spondylitis, axial ankylosing spondylitis, psoriasis, sweat gland abscess, ulcerative colitis, Crohn's disease, multiple sclerosis (MS), psoriatic arthritis, asthma, lupus (SLE), and sepsis (see, for example, Aggarwal et al., J. Leukoc. Biol., 71(1): 1-8 (2002); Lubberts et al., "Treatment with a neutralizing anti-murine interleukin-17 antibody after the onset of collagen-induced arthritis reduces joint inflammation, cartilage destruction, and bone erosion," Arthritis Rheum., 50: 650-659 (2004)).

[0102] In some embodiments, the multispecific antibody described herein binds to the amino acid sequence of the IL-17 family or a portion thereof. In some embodiments, the multispecific antibody described herein binds to the amino acid sequence of the IL-17A family or a portion thereof. In some embodiments, the multispecific antibody described herein binds to the amino acid sequence of the IL-17A / F family or a portion thereof. In some embodiments, the multispecific antibody described herein binds to the amino acid sequence of the IL17R family or a portion thereof. In some embodiments, the multispecific antibody contains a heavy chain that binds to at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of IL-17 type amino acids. In some embodiments, the multispecific antibody contains a light chain that binds to at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of IL-17 type amino acids. Embodiments coupled to IL-17B, IL-17C, IL-17D, IL-17E, and / or IL-17F are also within the scope of this disclosure.

[0103] In some embodiments, the IL-17 cytokine family functions as a heterodimer with proteins of the IL-17R family. In some embodiments, IL-17R includes IL-17RA, IL-17RB, IL-17RC, IL-17RD, IL-17RE, IL-17RF, or a combination thereof. Therefore, in some embodiments, the multispecific antibodies described herein can bind to any one of IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, IL-17F, IL-17RA, IL-17RB, IL-17RC, IL-17RD, IL-17RE, or a combination thereof.

[0104] In some embodiments, the multispecific antibody described herein comprises one of the combinations of CDR-H or its variants listed in Table 2, wherein the multispecific antibody can bind to IL-17, IL-17R or a combination thereof. In some embodiments, the CDR-H variant comprises at least one, two, or three substitutions, deletions, additions, or combinations thereof compared to the corresponding parent CDR-H sequence listed in Table 2. In some embodiments, the CDR-H or its variant comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the corresponding parent CDR-H sequence listed in Table 2. [Table 2]

[0105] In some embodiments, the multispecific antibodies described herein comprise an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to any one of the VH sequences listed in Table 3, wherein the multispecific antibody can bind to IL-17 family cytokines, IL-17R, or a combination thereof. In some embodiments, the multispecific antibodies described herein comprise an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to any one of the VH sequences listed in Table 3, wherein the multispecific antibody can bind to IL-17 family cytokines, IL-17R, or a combination thereof, and TREM1. [Table 3-1] [Table 3-2]

[0106] In some embodiments, the multispecific antibody described herein comprises one of the combinations of CDR-L or its variants listed in Table 4, wherein the multispecific antibody can bind to TREM1. In some embodiments, the CDR-L variant comprises at least one, two, or three substitutions, deletions, additions, or combinations thereof compared to the corresponding parent CDR-L sequence listed in Table 4. In some embodiments, the CDR-L or its variant comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the corresponding parent CDR-L sequence listed in Table 4. [Table 4]

[0107] In some embodiments, the multispecific antibodies described herein include combinations of CDRs, wherein the CDR comprises CDR-H1 or a variant thereof, CDR-H2 or a variant thereof, CDR-H3 or a variant thereof, CDR-L1 or a variant thereof, CDR-L2 or a variant thereof, and CDR-L3 or a variant thereof, and the combination is one of the combinations provided in Table 5. [Table 5]

[0108] In some embodiments, the multispecific antibodies described herein comprise an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to any one of the VL sequences listed in Table 6, wherein the multispecific antibody can bind to TREM1. [Table 6]

[0109] In some embodiments, the multispecific antibodies described herein include (a) a VH sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to any one of the amino acid sequences listed in Table 3; and (b) a VL sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to any one of the amino acid sequences listed in Table 6, wherein the multispecific antibody can bind to TREM1 and includes the VH and VL sequences in the combinations listed in Table 7. [Table 7]

[0110] In some embodiments, the multispecific antibodies described herein comprise a heavy chain (HC) or a variant thereof, wherein the HC comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the corresponding parental HC sequence described in Table 14. [Table 14-1] [Table 14-2]

[0111] In some embodiments, the multispecific antibodies described herein include a light chain (LC) or a variant thereof, wherein the LC comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the corresponding parental HC sequence listed in Table 15. [Table 15]

[0112] In some embodiments, the multispecific antibodies described herein include (a) an HC sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to any one of the amino acid sequences listed in Table 14; and (b) an LC sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to any one of the amino acid sequences listed in Table 15, wherein the multispecific antibody can bind to TREM1 and includes VH and VL sequences in combinations listed in Table 16. [Table 16] TREM1

[0113] The provocation receptor 1 (TREM1, also known as CD354, HGNC:17760, Entrez Gene:54210, UniProtKB:Q9NP99), expressed on myeloid cells, belongs to the Ig superfamily of receptors and is highly expressed on a subset of myeloid cells, including neutrophils, monocytes, and macrophages. TREM1 is a cell surface receptor involved in innate and adaptive immune functions by amplifying inflammatory responses. Instead of containing its own signaling motif, TREM1 receptor activation is mediated by the adapter DAP12 (DNAX-activated protein 12), which can lead to amplification of inflammatory responses (Bouchon, et al (2000) J. Immunol. 164 (10): 4991-4995). TREM1 crosslinking induces the expression of IL-8, myeloperoxidase, TNFα, and MCP-1, and TREM1 expression can be upregulated on myeloid cells in response to Toll-like receptor (TLR) stimulation (bacterial and fungal stimulation). TREM1 expression has also been shown to contribute to and amplify the acute inflammatory response between septic shock and infection (Cohen, (2001) Lancet. 358: 776-778). Furthermore, TREM1 has been associated with other diseases, including, but not limited to, IBD (UC and Crohn's disease), NEC, RA, PsO, nephritis, and SLE, as well as sepsis (see Colonna, M. The biology of TREM receptors. Nat Rev Immunol (2023). https: / / doi.org / 10.1038 / s41577-023-00837-1). In some embodiments, PGLYRP1 (peptidoglycan-recognizing protein 1) is targeted by the molecules described herein to bind to TREM1. Five activated forms of the TREM receptor exist, including TREM1, 2, 3, 4, and 5, with the soluble form of TREM1 (sTREM1) being released during infection. TREM1 consists of a single type V immunoglobulin (Ig)-like domain (Ig-V) of approximately 108 amino acids, followed by a stem region of 70 amino acids. In some embodiments, the molecules described herein bind to sTREM1 or a portion thereof.In some cases, the bispecific molecules described herein bind to specific domains in TREM1 or sTREM1, such as the Ig-V domain or the stem region.

[0114] In some embodiments, the multispecific antibodies described herein can bind to the TREM1 protein, which contains an antibody that inactivates unstimulated myeloid cells. In some embodiments, the multispecific (e.g., bispecific, tripspecific) molecules described herein bind to the mammalian TREM1 sequence. In some cases, TREM1 is a mouse homolog.

[0115] In some embodiments, the molecules described herein bind to a human TREM1 protein having one of the sequences listed in Table 26. [Table 26]

[0116] In some embodiments, the multispecific antibody described herein comprises one of the CDR-H or variants thereof listed in Table 8, wherein the multispecific antibody can bind to TREM1. In some embodiments, the CDR-H variant comprises at least one, two, or three substitutions, deletions, additions, or combinations thereof compared to the corresponding parent CDR-H sequence listed in Table 8. In some embodiments, the CDR-H or variant comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the corresponding parent CDR-H sequence listed in Table 8. [Table 8-1] [Table 8-2] [Table 8-3]

[0117] In some embodiments, the multispecific antibody described herein comprises one of the combinations of CDR-H or its variants listed in Table 27, wherein the multispecific antibody can bind to TREM1. [Table 27-1] [Table 27-2]

[0118] In some embodiments, the multispecific antibodies described herein comprise an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to any one of the VH sequences listed in Table 9, wherein the multispecific antibody can bind to TREM1. In some embodiments, the multispecific antibodies described herein comprise an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to any one of the VH sequences listed in Table 9, wherein the multispecific antibody can bind to TREM1 and a cytokine of the IL-17 family, IL-17R, or a combination thereof. [Table 9-1] [Table 9-2] [Table 9-3] [Table 9-4] [Table 9-5] [Table 9-6] [Table 9-7]

[0119] In some embodiments, the multispecific antibody described herein comprises one of the CDR-L or variants thereof listed in Table 10, wherein the multispecific antibody can bind to TREM1. In some embodiments, the CDR-L variant comprises at least one, two, or three substitutions, deletions, additions, or combinations thereof compared to the corresponding parent CDR-L sequence listed in Table 10. In some embodiments, the CDR-L or variant comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the corresponding parent CDR-L sequence listed in Table 10. [Table 10-1] [Table 10-2] [Table 10-3]

[0120] In some embodiments, the multispecific antibody described herein comprises one of the combinations of CDR-L or its variants listed in Table 28, wherein the multispecific antibody can bind to TREM1. [Table 28]

[0121] In some embodiments, the multispecific antibodies described herein include combinations of CDRs, wherein the CDR comprises CDR-H1 or a variant thereof, CDR-H2 or a variant thereof, CDR-H3 or a variant thereof, CDR-L1 or a variant thereof, CDR-L2 or a variant thereof, and CDR-L3 or a variant thereof, and the combination is one of the combinations provided in Table 11. [Table 11]

[0122] In some embodiments, the multispecific antibodies described herein comprise an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to any one of the VL sequences listed in Table 12, wherein the multispecific antibody can bind to TREM1. [Table 12-1] [Table 12-2] [Table 12-3] [Table 12-4] [Table 12-5] [Table 12-6]

[0123] In some embodiments, the multispecific antibodies described herein include (a) a VH sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to any one of the amino acid sequences listed in Table 9; and (b) a VL sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to any one of the amino acid sequences listed in Table 12, wherein the multispecific antibody can bind to TREM1 and includes the VH and VL sequences in the combinations listed in Table 13. [Table 13-1] [Table 13-2]

[0124] In some embodiments, the multispecific antibodies described herein comprise a heavy chain (HC) or a variant thereof, wherein the HC comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the corresponding parental HC sequence described in Table 17. [Table 17-1] [Table 17-2]

[0125] In some embodiments, the multispecific antibodies described herein include a light chain (LC) or a variant thereof, wherein the LC includes an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the corresponding parental HC sequence listed in Table 18. [Table 18]

[0126] In some embodiments, the multispecific antibodies described herein include (a) an HC sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to any one of the amino acid sequences listed in Table 17; and (b) an LC sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to any one of the amino acid sequences listed in Table 18, wherein the multispecific antibody can bind to TREM1 and includes VH and VL sequences in combinations listed in Table 19. [Table 19] Nucleotide constructs for IL17 and / or TREM1 targeting proteins

[0127] Nucleotide sequences for targeting / binding to IL-17 family cytokines, IL-17R, or combinations thereof are provided herein. In some embodiments, the nucleotide sequence encodes any HC that binds to IL-17 family cytokines, IL-17R, or combinations thereof. In some embodiments, the HC comprises one of the CDR-H or variant combinations listed in Table 2, wherein the variant comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the corresponding parental CDR-H sequence listed in Table 2. In some embodiments, the HC comprises one of the VH sequences or variants listed in Table 3, wherein the variant comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the corresponding parental VH sequence listed in Table 3. In some embodiments, the HC comprises one of the CDRs or variants thereof listed in Table 4, wherein the variant comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the corresponding parent CDR sequence listed in Table 4. In some embodiments, the HC comprises one of the HC sequences or variants thereof listed in Table 14, wherein the variant comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the corresponding parent HC sequence listed in Table 14.

[0128] Nucleotide sequences for targeting / binding to IL-17 family cytokines, IL-17R, or combinations thereof are provided herein. In some embodiments, the nucleotide sequence encodes any LC that binds to IL-17 family cytokines, IL-17R, or combinations thereof. In some embodiments, the LC comprises one of the CDR-L or variant combinations listed in Table 4, wherein the variant comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the corresponding parent CDR-L sequence listed in Table 4. In some embodiments, the LC comprises one of the VL sequences or variants listed in Table 6, wherein the variant comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the corresponding parent VL sequence listed in Table 6. In some embodiments, the LC comprises one of the combinations of CDR or variants thereof listed in Table 4, wherein the variant comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the corresponding parent CDR sequence listed in Table 4. In some embodiments, the LC comprises one of the LC sequences or variants thereof listed in Table 15, wherein the variant comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the corresponding parent LC sequence listed in Table 15.

[0129] The nucleotide sequence encodes any HC that binds to TREM1. In some embodiments, the HC comprises one of the combinations of CDR-H or its variants listed in Table 27, wherein the variant comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the corresponding parental CDR-H sequence listed in Table 8. In some embodiments, the HC comprises one of the VH sequences or its variants listed in Table 9, wherein the variant comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the corresponding parental VH sequence listed in Table 9. In some embodiments, the HC comprises one of the combinations of CDR or variants thereof listed in Table 27, wherein the variant comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the corresponding parent CDR sequence listed in Table 8. In some embodiments, the HC comprises one of the HC sequences or variants thereof listed in Table 17, wherein the variant comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the corresponding parent HC sequence listed in Table 17.

[0130] The nucleotide sequence encodes any LC that binds to TREM1. In some embodiments, the LC comprises one of the combinations of CDR-L or its variants listed in Table 28, wherein the variant comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the corresponding parent CDR-L sequence listed in Table 10. In some embodiments, the LC comprises one of the VL sequences or its variants listed in Table 12, wherein the variant comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the corresponding parent VL sequence listed in Table 12. In some embodiments, the LC comprises one of the combinations of CDR or variants thereof listed in Table 28, wherein the variant comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the corresponding parent CDR-L sequence listed in Table 10. In some embodiments, the LC comprises one of the LC sequences or variants thereof listed in Table 18, wherein the variant comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the corresponding parent LC sequence listed in Table 18.

[0131] Table 20 provides exemplary nucleotide sequences of some of the proteins or parts thereof described herein. In some embodiments, the nucleotide sequences encoding BsAb or parts thereof include sequences that are at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to any one of the nucleotide sequences listed in Table 20. [Table 20-1] Table 20-2 Table 20-3 Table 20-4 Table 20-5 Table 20-6 Table 20-7 Table 20-8 Table 20-9 BsAb

[0132] Bispecific antibodies that bind to TREM1 and the IL-17 family cytokine IL-17R or a combination thereof are disclosed herein. In some embodiments, the BsAb includes a heavy chain region. In some embodiments, the BsAb includes a light chain region. In some embodiments, the light chain region includes a kappa light chain constant region. In some embodiments, the BsAb includes a Bsab light chain variable region. In some embodiments, the BsAb includes a Bsab heavy chain variable region. In some embodiments, the BsAb includes a Bsab light chain variable region and an IgG heavy chain variable region. In some embodiments, the BsAb may be a humanized antibody. In some embodiments, the BsAb may be a chimeric antibody. In some embodiments, the BsAb may be a human antibody. In some embodiments, the BsAb includes a common light chain (L chain). In some embodiments, the L chain acts against a specific antigen. The use of a common light chain may allow for preferential heterodimerization in the Fc region. In some embodiments, the L chain includes a knob mutation. In some embodiments, the L chain contains a hole mutation. In some embodiments, preferential heterodimer formation is preferred when Bsab containing a common L chain is bound. In some embodiments, glutathione disulfide exchange is used to assemble the heterodimer pair.

[0133] In some embodiments, BsAb may be a monospecific antibody, but is not limited to, an antibody in which both arms target different epitopes of the same antigen. In some embodiments, BsAb may be a bispecific antibody. In some embodiments, BsAb may be a triplicate antibody. In some embodiments, BsAb may be a multispecific antibody.

[0134] In some embodiments, the BsAbs described herein induce degradation of TREM1, cleavage of TREM1, internalization of TREM1, desorption of TREM1, downregulation of TREM1 expression, or a combination thereof. In some embodiments, the BsAbs described herein inhibit the interaction (e.g., binding) between TREM1 and one or more TREM1 ligands. In some embodiments, the BsAbs described herein transiently activate and then induce one or more of the following: degradation of TREM1, cleavage of TREM1, internalization of TREM1, desorption of TREM1, downregulation of TREM1 expression, or reduction of TREM1 expression.

[0135] In some embodiments, the BsAbs described herein bind to inflammatory receptors and inflammatory cytokines. Inflammatory receptors amplify the immune response. For example, TREM1 is an inflammatory receptor that, upon activation, induces the production of inflammatory cytokines such as IL-1β, IL-2, IL-6, IL-8, IL-12p40, and TNF-α; chemokines such as MIP-1α, membrane cofactor proteins-1 and -2, and GM-CSF; and costimulatory molecules such as CD1a, CD86, and MHC class II. Therefore, TREM1 is associated with the development of immune-associated inflammatory diseases. A decrease in TREM1 activity may result in a decrease / attenuation of IL-17 production. Therefore, in some embodiments, treatment with the BsAb described herein favorably reduces the IL-17 inflammatory effect through two independent mechanisms: (1) direct inhibition of IL-17 activity by binding to IL-17, and (2) indirect inhibition of IL-17 activity by reducing IL-17 production by binding to TREM1.

[0136] In some embodiments, administration of the BsAb described herein, which includes an IL-17 binding domain, results in a decrease in TREM-1 expression in subjects with an autoimmune state. Therefore, in some embodiments, administration of the BsAb described herein to subjects with an autoimmune state can favorably reduce TREM1 activity through two independent mechanisms: (1) direct inhibition of TREM1 activity by binding to TREM1, and (2) indirect inhibition of TREM1 activity by reducing TREM1 expression through a decrease in IL-17 activity.

[0137] In some embodiments, administration of the BsAb described herein, which includes a TREM-1 binding domain, results in increased expression of TREM-1 related genes. In some embodiments, the TREM-1 related genes include nicotinamide phosphoribosyltransferase (NAMPT), dehydrogenase / reductase 9 (DHRS9), cyclin-dependent kinase inhibitor 1A (CDKN1A), CD52 molecule (CD52), myotubularin-related protein 11 (MTMR11), EH domain-containing protein 1 (EHD1), solute transporter family 27 member 3 (SLC27A3), interleukin 24 (IL24), Pim-2 oncogene serine / threonine kinase (PIM2), chitinase 3-like protein 1 (CHI3L1), polypeptide N-acetylgalactosaminyl This includes lanceferase 6 (GALNT6), acyl-CoA thioesterase 7 (ACOT7), cytokine-inducible SH2-containing protein (CISH), family containing sequence similarity 129 member A (FAM129A), polo-like kinase 3 (PLK3), major facilitator superfamily domain-containing 12 (MFSD12), StAR-related lipid transfer domain-containing 4 (STARD4), c-type lectin domain family 12 member A (CLEC12A), CD55 molecule (Cromer blood group) (CD55), interferon lambda receptor 1 (IFNLR1), or combinations thereof.

[0138] High activity of TREM1 in peripheral myeloid cells and / or microglia may contribute to age-related inflammation and cognitive decline. TREM1 represses the expression of genes associated with key enzymes in the pentose phosphate pathway, subsequently creating a deficiency in ribose-5-phosphate, an essential intermediate for purine and pyrimidine synthesis. This deficiency can be corrected by reducing TREM1 activity. Therefore, in some embodiments, administration of the BsAb described herein, which contains a TREM-1 binding domain, restores the pentose phosphate pathway (PPP). In some embodiments, administration of the BsAb described herein, which contains a TREM-1 binding domain, restores the expression of hexokinase, a transcript of glycolysis, and the efficiency of pyruvate kinase. In some embodiments, administration of the BsAb described herein prevents cognitive decline. Furthermore, TREM1 expression / activity is also associated with amyloid and tau pathology in subjects with Alzheimer's disease. Therefore, in some embodiments, the BsAb described herein contains neuroprotective activity.

[0139] In some embodiments, the BsAb described herein comprises anti-inflammatory activity at least equivalent to that of a combination of a monospecific antibody that binds to TREM1 and a monospecific antibody that binds to a cytokine of the IL-17 family, IL-17R, or a combination thereof. In some embodiments, the BsAb advantageously reduces the need to administer two separate antibodies. In some embodiments, the subject treated with the BsAb advantageously tolerates higher doses compared to a combination of monospecific antibodies.

[0140] In some embodiments, the BsAbs described herein contain at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, or higher anti-inflammatory activity compared to a combination of a monospecific antibody that binds to TREM1 and a monospecific antibody that binds to a cytokine of the IL-17 family, IL-17R, or a combination thereof.

[0141] Furthermore, the BsAbs described herein contain two therapeutic domains (a TREM1-binding domain and an IL-17-binding domain) for all Fc regions. In contrast, monospecific antibodies contained one therapeutic domain (either a TREM1-binding domain or an IL-17-binding domain) for all Fc regions. Therefore, administration of BsAbs advantageously reduces the amount of Fc region administered. In some embodiments, BsAbs advantageously exhibit lower adverse effects compared to the administration of two separate antibodies. In some embodiments, the BsAbs described herein advantageously contain higher anti-inflammatory activity compared to the combined anti-inflammatory activity of a monospecific antibody that binds to TREM1 and a monospecific antibody that binds to the cytokine IL-17R or a combination thereof from the IL-17 family.

[0142] Treatment with IL-17-binding antibodies to reduce the IL-17-mediated inflammatory response in subjects may cause upper respiratory tract infections, headache, nausea, vomiting, rectal bleeding, sore throat, Candida infections, hypersensitivity reactions, hypertension, diarrhea, back pain, cough, malignancies, and major adverse cardiovascular events. For example, inhibition of IL-17 activity in subjects to treat psoriasis may cause oral candidiasis. Therefore, administration of the BsAb described herein to subjects to treat an inflammatory condition (or symptom) will experience a lower incidence (or occurrence) of one or more adverse effects associated with the reduction of IL-17 activity compared to subjects treated with a monospecific IL-17-binding antibody that reduces IL-17 activity. For example, in some embodiments, treatment of psoriasis in subjects with the BsAb described herein reduces the incidence of Candida infections in subjects compared to subjects treated with a monospecific antibody that binds to and / or reduces the activity of IL-17 family cytokines.

[0143] In some embodiments, the BsAb described herein is engineered to include pH-dependent target-binding activity. In some embodiments, the BsAb includes such engineered BsAb. In some embodiments, the engineered BsAb includes pH-dependent target-binding activity with respect to at least one target, where the at least one target is selected from TREM1, cytokines of the IL-17 family, and IL-17R. In some embodiments, the engineered BsAb readily binds to at least one target at neutral pH and dissociates from at least one target at acidic pH. Thus, the engineered BsAb binds to at least one target in plasma with neutral pH and dissociates from at least one target in endosomes with acidic pH. The dissociation of the engineered BsAb from at least one target in endosomes allows for the recycling of the engineered BsAb into plasma via FcRn. However, at least one target is transported to lysosomes and degraded. Such properties of the engineered BsAb allow for the removal of at least one target from plasma. Thus, in some embodiments, the engineered BsAb possesses target removal activity with respect to at least one target, where the at least one target is selected from TREM1, cytokines of the IL-17 family, and IL-17R.

[0144] In some embodiments, the BsAb described herein targets / binds to IL17 and TREM1 and / or sTREM1. For example, in some embodiments, the BsAb comprises a first HC targeting IL17 and a second HC targeting TREM1 and / or sTREM1. In some embodiments, the first HC comprises one of the combinations of CDR-H or its variants listed in Table 2, wherein the variant comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the corresponding parent CDR-H sequence listed in Table 2. In some embodiments, the first HC comprises one of the VH sequences or variants thereof listed in Table 3, wherein the variant comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the corresponding parent VH sequence listed in Table 3. In some embodiments, the first HC comprises one of the combinations of CDRs or variants thereof listed in Table 4, wherein the variant comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the corresponding parent CDR sequence listed in Table 4. In some embodiments, the first HC comprises one of the HC sequences or variants thereof listed in Table 14, wherein the variant comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the corresponding parent HC sequence listed in Table 14.

[0145] In some embodiments, the BsAb described herein targets and binds to IL17 and TREM1 and / or sTREM1, wherein the BsAb comprises a first HC targeting IL17 and a second HC targeting TREM1 and / or sTREM1. In some embodiments, the second HC comprises one of the combinations of CDR-H or its variants listed in Table 8, wherein the variant comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the corresponding parent CDR-H sequence listed in Table 8. In some embodiments, the second HC comprises one of the VH sequences or variants thereof listed in Table 9, wherein the variant comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the corresponding parent VH sequence listed in Table 9. In some embodiments, the second HC comprises one of the combinations of CDRs or variants thereof listed in Table 11, wherein the variant comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the corresponding parent CDR sequence listed in Table 11. In some embodiments, the second HC comprises one of the HC sequences or variants thereof listed in Table 17, wherein the variant comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the corresponding parent HC sequence listed in Table 17.

[0146] In some embodiments, the BsAb described herein includes a first LC, a second LC, or a combination thereof. Thus, in some embodiments, the first LC includes any one of the combinations of CDR-L or its variants listed in Table 4, wherein the variant includes an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the corresponding parent CDR-L sequence listed in Table 4. In some embodiments, the first LC includes any one of the VL sequences or its variants listed in Table 6, wherein the variant includes an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the corresponding parent CDR-L sequence listed in Table 6. In some embodiments, the first LC comprises one of the combinations of CDRs or variants thereof listed in Table 5, wherein the variant comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the corresponding parent CDR sequence listed in Table 5. In some embodiments, the first LC comprises one of the LC sequences or variants thereof listed in Table 15, wherein the variant comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the corresponding parent LC sequence listed in Table 15.

[0147] In some embodiments, the BsAb described herein comprises a first LC, a second LC, or a combination thereof. In some embodiments, the second LC comprises one of the combinations of CDR-L or its variants listed in Table 10, wherein the variant comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the corresponding parent CDR-L sequence listed in Table 10. In some embodiments, the second LC comprises one of the VL sequences or its variants listed in Table 12, wherein the variant comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the corresponding parent CDR-L sequence listed in Table 12. In some embodiments, the second LC comprises one of the combinations of CDR or variants thereof listed in Table 11, wherein the variant comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the corresponding parent CDR sequence listed in Table 11. In some embodiments, the second LC comprises one of the LC sequences or variants thereof listed in Table 18, wherein the variant comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the corresponding parent LC sequence listed in Table 18.

[0148] Exemplary BsAb candidates are provided in Table 22. In some embodiments, the BsAb described herein comprises a first HC, a second HC, and at least one LC or a variant thereof, wherein the variant comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the corresponding parent amino acid sequence described in Table 22. [Table 22]

[0149] In some embodiments, the bispecific antibody described herein includes at least one constant region. In some embodiments, the bispecific antibody described herein includes two constant regions. In some embodiments, the two constant regions are derived from the human IgG1 heavy chain constant chain. In some embodiments, the two constant regions are a first constant region and a second constant region. In some embodiments, the first constant region is manipulated to include a knob, and the second constant region is manipulated to include a hole. Thus, in some embodiments, the first constant region includes a mutation at position T366 in EU numbering, and the second constant region includes a mutation at position Y407 in EU numbering. In some embodiments, the first constant region includes a mutation at position T366 in EU numbering, and the second constant region includes mutations at positions T366 and Y407 in EU numbering. In some embodiments, the first constant region includes a mutation at position T366 in EU numbering, and the second constant region includes mutations at positions T366, L368, and Y407 in EU numbering. In some embodiments, the first constant region includes an S354C mutation, a T366W mutation, or a combination thereof in EU numbering, and the second constant region includes a Y349C mutation, a T366S mutation, a Y407V mutation, or a combination thereof in EU numbering.

[0150] Alternatively, in some embodiments, the bispecific antibodies described herein comprise two constant regions derived from a single human IgG constant chain, wherein the two constant regions comprise a first constant region and a second constant region, the first constant region comprising at least two mutations, and the second constant region comprising at least one mutation. For example, in some embodiments, the first constant region comprises at least two mutations selected from EU numbering positions L351, F405, and Y407, and the second constant region comprises at least one mutation selected from EU numbering positions T366, K392, and T394. In some embodiments, the mutation at L351 comprises L351Y and L351A substitutions. In some embodiments, the mutation at F405 comprises F405A, F405S, F405T, and F405V substitutions. In some embodiments, mutations at position Y407 include Y407A, Y407V, Y407S, and Y407I substitutions. In some embodiments, mutations at position T366 include T366L, T366M, T366V, and T366I substitutions. In some embodiments, mutations at position K392 include K392C, K392M, K392L, K392I, K392E, K392D, and K392F substitutions. In some embodiments, mutations at position T394 include T394D, T394W, T394V, and T394S substitutions. In some embodiments, at least two mutations in the first constant region further include one or more mutations at positions Q347, Y349, T350, K370, G371, D399, and S400 in EU numbering. In some embodiments, mutations at position Q347 include Q347R, Q347E, and Q347K substitutions. In some embodiments, mutations at position Y349 include Y349C substitutions. In some embodiments, mutations at position T350 include T350V substitutions. In some embodiments, mutations at position K370 include K370T substitutions. In some embodiments, mutations at position G371 include G371D and G371S substitutions. In some embodiments, mutations at position D399 include D399C, D399R, and D399K substitutions. In some embodiments, mutations at position S400 include S400D, S400K, S400E, and S400R substitutions.In some embodiments, at least two mutations in the second constant region further include one or more mutations at EU numbering positions T350, S354, E357, K360, Q362E, S364, N390, K409, and T411. In some embodiments, the mutation at T350 includes a T350V substitution. In some embodiments, the mutation at S354 includes an S354C substitution. In some embodiments, the mutation at E357 includes an E357Q substitution. In some embodiments, the mutation at K360 includes K360D and K360E substitutions. In some embodiments, the mutation at Q362 includes a Q362E substitution. In some embodiments, the mutation at S364 includes an S364R substitution. In some embodiments, mutations at position N390 include N390K, N390R, N390D, and N390E substitutions. In some embodiments, mutations at position K409 include K409L, K409M, K409F, and K409W substitutions. In some embodiments, mutations at position T411 include T411R, T411D, T411I, T411K, T411E, T411N, T411S, and T411L substitutions.

[0151] Alternatively, in some embodiments, the bispecific antibody described herein comprises two constant regions derived from a single human IgG constant chain, wherein the two constant regions comprise a first constant region and a second constant region, and the first and second constant regions are manipulated to interact electrostatically with each other. In some embodiments, the first constant region comprises a substitution at EU numbering K370, having a negatively charged amino acid residue (e.g., aspartic acid, glutamic acid), and the second constant region comprises a substitution at EU numbering E357, having a positively charged amino acid residue (e.g., arginine, lysine, histidine). In some embodiments, the first constant region includes a substitution at EU numbering K392 or K409 having a negatively charged amino acid residue (e.g., aspartic acid, glutamic acid), and the second constant region includes a substitution at EU numbering D399 having a positively charged amino acid residue (e.g., arginine, lysine, histidine). In some embodiments, the first constant region includes a substitution at EU numbering K439 having a negatively charged amino acid residue (e.g., aspartic acid, glutamic acid), and the second constant region includes a substitution at EU numbering D356 having a positively charged amino acid residue (e.g., arginine, lysine, histidine).

[0152] In some embodiments, treatment with the BsAbs described herein reduces the level of at least one inflammatory marker (e.g., biomarker) in the serum of the subject by at least 10%, at least 20%, at least 30%, at least 40%, and at least 50% compared to treatment with the corresponding combination of two monospecific antibodies. Thus, treatment with BsAbs that bind to TREM1 and IL-17 reduces the level of at least one biomarker in the serum of the subject by 10% or more compared to treatment with the corresponding combination of a monospecific antibody targeting TREM1 and a monospecific antibody targeting IL-17. In some embodiments, at least one biomarker is matrix metalloproteinase 1 protein (MMP1), matrix metalloproteinase 2 protein (MMP2), matrix metalloproteinase 7 protein (MMP7), matrix metalloproteinase 10 protein (MMP10), granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor-alpha (TNFα), tumor necrosis factor superfamily member 15 (TNFSF15), IL-17, IL-1α, IL-1β, IL-6, IL-8, It contains IL-12, IL-23 subunit p19, IL-24, IL-36γ, IL-1RA, monocyte chemoattractant protein-1 (MCP-1), chemokine (CC motif) ligand 1 (CCL1), chemokine (CC motif) ligand 3 (CCL3), chemokine (CC motif) ligand 20 (CCL20), chitinase-3-like protein 1 (CHI3L1), prostaglandin-endoperoxidase synthase 2 (PTGS2), secretogranin V (SCG5), inhibin beta-A (INHBA), osteoclast-stimulating membrane protein (OCSTA MP), tissue factor pathway inhibitor 2 (TFPI2), coagulation factor III, G protein signaling regulator 16 (RGS16), and TREM1. BiTE antibody

[0153] In some embodiments, the molecules described herein include a bispecific T cell engager (BiTE) antibody construct, which may be referred to herein as the “BiTE molecule.” The BiTe antibody construct is a type of fusion protein. In some embodiments, the BiTE molecule activates T cell activity. In some embodiments, the BiTE molecule comprises two functional single-chain variable fragments. In some embodiments, the BiTE molecule comprises two binding domains. In some embodiments, the bispecific antibody described herein comprises a TREM1 binding domain. In some embodiments, the bispecific antibody described herein further comprises an interleukin binding domain. In some embodiments, the bispecific antibody comprises an engineered functional fragment of the TREM1 binding domain. In some embodiments, the bispecific antibody comprises an engineered functional fragment of the IL-17 family binding domain. In some embodiments, the bispecific antibody comprises an engineered functional fragment of the IL-17A binding domain. In some embodiments, the bispecific antibody comprises an engineered functional fragment of the IL-17A / F binding domain. In some embodiments, the bispecific antibody comprises an engineered functional fragment of the IL-17R binding domain. In some embodiments, the bispecific antibody includes a variant of the TREM1-binding domain. In some embodiments, the bispecific antibody includes a variant of the IL-17 family-binding domain. chemical crosslinking

[0154] The use of chemical crosslinking reagents to covalently bond two antibodies is well known in the industry. Functional antibody fragments, produced from their respective parent antibodies by enzymatic digestion or by recombinant technology, can be conjugated using bifunctional reagents (Glennie MJ et al., J Exp Med 1992; 175:217-225). Quadroma

[0155] Quadromes and triomas can be generated by fusing two hybridomas or one hybridoma with B lymphocytes, respectively (Suresh MR et al., Methods Enzymol 1986; 121: 210-228). In this case, the simultaneous expression of two heavy chains and two light chains results in a random assembly of 10 antibody combinations, with the desired bsAb being only a fraction of the secreted antibodies. The bsAb can be purified using a combination of chromatographic techniques. Recombinant multi-specificity antibodies

[0156] Most multispecific antibody forms can be generated by genetic engineering techniques that use functional antibody fragments, such as scFv or Fab fragments, as building blocks linked by polypeptide linkers. Forms based on linked functional antibody fragments include tandem scFv (BiTE), diabody, and tandem diabody (Kipriyanov SM. Methods Mol Biol 2003; 207:323-333; Korn T et al, Int J Cancer 2002; 100:690-697). These forms include diabody-Fc, tandem diabody-Fc, tandem diabody-CH3, (scFv)4-Fc, and DVD-Ig (Lu D et al, J Immunol Methods 2003; 279: 219-232; Lu D et al, J Biol Chem 2005; 280: 19665-19672; Lu D et al, J Biol Chem 2004; 279: 2856-2865; Wu C et al., Nat Biotechnol 2007 25: 1290-7). In some embodiments, the multispecific antibody is Fab2 antibody, bis-scFv antibody, diabody, DVD-Ig, TandAb, tandem scFv-Fc, 1-arm tandem scFv-Fc, DART, DART-Fc, or a functional fragment thereof. Further types of multispecific antibodies and their constructions are disclosed in Brinkmann & Kontermann (2017), mAbs, 9:2, 182-212, DOI: 10.1080 / 19420862.2016.1268307, and their contents are incorporated by reference.

[0157] Strategies based on the forced heterodimerization of two heavy chains are being explored. The first method to create a "knob-into-hole" relationship aims to force the pairing of two different IgG heavy chains by introducing mutations into the CH3 domain and modifying the contact interface (Ridgway JB et al., Protein Eng 1996; 9: 617-621). A "knob" was created by introducing an amino acid with a large side chain onto one chain. Conversely, a "hole" was created in the other CH3 domain by replacing the bulky amino acid with an amino acid with a short side chain. By simultaneously expressing these two heavy chains, heterodimerization ("knob-hole") was observed in over 90% of cases compared to homodimerization ("hole-hole" or "knob-knob"). A similar concept was developed using human CH3 domains with strand exchange manipulation domains (SEEDs) based on human IgG and human IgA sequences (Davis JH et al., 2010, PEDS 23: 195-202). These manipulated domains result in the formation of heterodimer molecules that can have two distinct specificities.

[0158] In recent years, an improvement over the "knob-into-hole" method, "CrossMab," has been described in WO2009 / 080253A1. This method involves the exchange of parts of the light and heavy chain domains in addition to the "knob-into-hole" mutation. Single-domain based antibodies

[0159] The immune systems of camelids (llamas and camels) and cartilaginous fish (whale sharks) utilize a single V domain fused to the Fc, demonstrating that a single domain can confer high affinity binding to an antigen. Camelid, shark, and even human V domains are antibody alternatives, but they are also used for bsAb production. They can be reformatted into classical IgGs, each arm having the ability to bind to two targets via its VH or VL domains.

[0160] The bispecific antibodies of this disclosure can be produced by any process disclosed in this application or otherwise known in the art. Dual variable domain immunoglobulin

[0161] Exemplary bispecific antibodies may contain individually encoded peptides or "segments" containing compact tertiary structures within a single continuous chain. The constituent peptides are selected to associate complementaryly, rather than asymmetrically to form homomultimers, and adopt a stable complex similar to the parent tertiary structure, so asymmetrically in their intended structure. At the gene level, these segments may be encoded by interchangeable cassettes having suitable restriction sites. These standardized cassettes can be fused via linkers or hinges to recombinant proteins with different C or N-terminuses in a suitable expression vector system. Polypeptide segments that do not have the ability to assemble as homodimers are induced by cleaving a parent polypeptide having a compact tertiary structure. These polypeptide segments can then be fused to one or more different functional domains at the gene level. These different polypeptide segments fused to one or more functional domains can then be co-expressed, for example, to result in the formation of a native-like parent structure bound to the functional domain. This parent structure is formed by the dimerization of polypeptide segments derived from the original parent polypeptide. The resulting multifunctional constructs appear as compact tertiary structures bound to one or more functional domains. Once the structural subdomains are identified, the protein is cleaved in such a way that these subdomains remain intact.As part of this disclosure, DNA sequences, vectors, preferably two-cistronic vectors, and vector cassettes can be prepared, characterized in that they include an amino acid sequence and optionally at least one further (poly)peptide contained in the multifunctional polypeptide of the present invention, and further, at least one, preferably single cloning site encoding DNA for insertion of DNA encoding at least one further functional domain, or they include an amino acid sequence and optionally a further (poly)peptide contained in the multifunctional polypeptide of the present invention and a DNA sequence encoding a suitable restriction site for cloning the DNA sequence encoding the functional domain. The vector cassette is characterized in that it includes a host cell transformed with the inserted DNA sequence encoding the functional domain and at least one vector or vector cassette of the present invention that can be used for the preparation of the bispecific or multifunctional polypeptide. The host cell may be a mammal, preferably human, yeast, insect, plant, or bacterium, preferably E. coli cell. A bispecific antibody can be prepared by a method comprising culturing at least two host cells of the present invention, each producing only one of the first and second amino acid sequences bound to at least one further functional domain, in a suitable medium; recovering the amino acid sequences; mixing them under mild denaturation conditions; and enabling in vitro folding of the multifunctional polypeptide of the present invention from the amino acid sequences. The method may be characterized in that the further amino acid sequence bound to at least one further functional domain is produced by at least one further host cell that does not produce the first or second amino acid sequence.Furthermore, the method may be characterized in that at least one further amino acid sequence bound to at least one further functional domain is produced by the host cell of the present invention that produces the first or second amino acid sequence.

[0162] If either the second or first portion of an antibody construct described herein contains two antibody-variable domains, these two antibody-variable domains may be VH and VL domains associated with each other. However, it is also intended that the two antibody-variable domains contained in either the second or first portion may be two VH domains or two VL regions associated with each other. In the case where the two antibody-variable domains of the first or second portion are co-associated with each other, the two antibody-variable domains can be designed as an scFv fragment, meaning that the two domains are separated from each other by a peptide linker of sufficient length to allow intermolecular association between these two domains. Suitable linker designs for this purpose are described in the prior art, for example, in the authorized patents EP623679B1, U.S. Patent No. 5,258,498, EP573551B1, and U.S. Patent No. 5,525,491. In other words, a bispecific antibody may be a construct having a total of three antibody-variable domains. One antibody-variable domain, alone, i.e., without pairing with another antibody-variable domain, specifically binds to either human immune effector cells or target cells by (a) specifically binding to effector antigens on human immune effector cells, while the remaining two antibody-variable domains (b) specifically bind together to either human immune effector cells or effector antigens on human immune effector cells, respectively. In this case, the presence of three antibody-variable domains in a bispecific antibody offers unique advantages. scFv exhibiting desirable binding specificity to target antigens are already known and optimized, and the omission of one of its two antibody-variable domains invalidates or at least weakens its binding properties. Such scFv may constitute part of the antibody constructs described herein. Specifically, such a three-domain antibody may advantageously include the entire scFv as either its effector antigen-donating portion or its target antigen-donating portion.Next, efficiently, this involves the simple incorporation of just one additional antibody-variable domain into the same polypeptide chain as the scFv to form a bispecific antibody starting from a desired scFv, where the incorporation of one additional antibody-variable domain has a different antigen-binding specificity than that of the scFv. The first and second portions of the bispecific antibody can be separated from each other by a synthetic polypeptide spacer portion, covalently (i.e., by a peptide bond) linking the C-terminus of the first portion to the N-terminus of the second portion, or the C-terminus of the second portion to the N-terminus of the first portion. Thus, these bispecific antibody portions can be arranged as either N-(first portion)-(second portion)-C or N-(second portion)-(first portion)-C. In some embodiments, the binding site for the second specificity is fused to the N or C-terminus of the heavy or light chain, for example, in the form of an scFv fragment or a variable single domain, to obtain a bispecific tetravalent molecule. The bispecific molecule produced by the fusion of the scFv fragment to the mAb results in high flexibility. Generally, scFv molecules can be ligated to the C-terminus as well as the N-terminus of the heavy or light chain variable domain of an mAb without impairing productivity or antigen-binding activity. This group of bispecific molecules also includes DVD-Ig, in which the second VH and VL domains are fused to the heavy and light chains, respectively; two-in-one antibodies, in which the second specificity is introduced to the native binding site of an IgG molecule; and mAb2 molecules, in which the second specificity is constructed within the CH3 domain of the Fc region. A defining characteristic of all these molecules is the symmetry resulting from the dimerized assembly of two identical heavy chains, an inherent property of these chains.

[0163] Heavy chain heterodimerization can be achieved by manipulating charged CH3 interfaces to introduce an electrostatic steering effect, or by using chain exchange manipulation domain technology (SEEDbody) with CH3 sequences composed of alternating segments derived from human IgA and IgG. In contrast to bispecific IgG-like molecules, these bispecific antibodies are bivalent and have essentially the same size as those of IgG. Fc heterodimerization has recently been applied to generate trivalent bispecific molecules (HA-TF Fc variants) by fusing VH and VL domains to the C-terminus of the manipulated heavy chain. By combining the variable domains of two antibodies, bispecific antibodies with molecular weights in the range of 50-100 kDa can be generated. For example, two scFvs were linked by a series-oriented, more or less flexible peptide linker (tandem scFv, taFv, tascFv), which can be further extended by additional scFvs, to generate, for example, bispecific or triplicate triple bodies (sctb). A diabody is a heterodimer molecule composed of variable domains of two antibodies arranged in either the VHA-VLB and VHB-VLA order (VH-VL orientation) or the VLA-VHB and VLB-VHA order (VL-VH orientation). The linker connecting the two domains within a single chain consists of approximately five residues, resulting in the formation of a head-tail assembly and thus a compact molecule with two functional binding sites after the simultaneous expression of the two chains within a single cell. The diabody (Db) form was further stabilized by introducing an interchain disulfide bond (dsDb, DART molecule) or by generating a single-chain derivative (scDb). Reducing the central linker can convert scDb into a tetravalent molecule, resulting in homodimerization of the two chains. Additionally, fusing scFv to the heavy or light chain of a Fab fragment produced small bispecific molecules. Furthermore, tetravalent derivatives were generated by fusing tandem scFv, diabody, and scDb to an Fc or CH3 domain.Furthermore, scFv can be combined with an Fc or CH3 domain to generate a tetravalent molecule, for example, by fusing scFv to the N and C-terminuses of an Fc fragment, or by using a knob-in-to-hole technique to generate a bivalent scFv-Fc or scFv-CH3 molecule. A different technique for generating bispecific antibodies of the present invention is the dock-and-lock method (DNL). Many of the established bispecific antibody forms can also be combined with additional proteins and components, such as drugs, toxins, enzymes, and cytokines, to enable dual targeting and delivery of the fusion partner. In addition, fusion to plasma proteins such as serum albumin or albumin-binding moieties can be applied to extend the plasma half-life of bispecific antibodies. Structure of a bispecific antibody

[0164] In one example, the bispecific antibody may be a binding protein comprising a first polypeptide chain containing VH1-(X1)n-VH2-C--(X2)n (wherein VH1 is a first heavy chain variable domain, VH2 is a second heavy chain variable domain, C is a constant domain, X1 represents a polypeptide linker, X2 represents an Fc region, and n is 0 or 1). In some embodiments, VH1 and VH2 in the binding protein may be heavy chain variable domains selected from the group consisting of mouse heavy chain variable domains, human heavy chain variable domains, CDR-implanted heavy chain variable domains, and humanized heavy chain variable domains. VH1 and VH2 may be able to bind to different antigens. C may be a heavy chain constant domain. For example, X1 is a linker peptide. For example, X1 is a linker listed herein. In some embodiments, X2 is an Fc region. In other embodiments, X2 is a variant Fc region. In some embodiments, VH1 can bind to a first antigen and VH2 can bind to a second antigen. In some embodiments, VH1 can bind to a second antigen and VH2 can bind to a first antigen.

[0165] In one example, the bispecific antibody may be a binding protein comprising a second polypeptide chain containing VL1-(X1)n-VL2-C--(X2)n (wherein VL1 is a first light chain variable domain, VL2 is a second light chain variable domain, C is a constant domain, X1 represents a polypeptide linker, X2 represents an Fc region, and n is 0 or 1). In some embodiments, VL1 and VL2 in the binding protein may be light chain variable domains selected from the group consisting of mouse light chain variable domains, human light chain variable domains, CDR-implanted light chain variable domains, and humanized light chain variable domains. VL1 and VL2 may be able to bind to different antigens. C may be a heavy chain constant domain. For example, X1 is a linker peptide. For example, X1 is a linker listed herein. In some embodiments, X2 is an Fc region. In other embodiments, X2 is a variant Fc region. In some embodiments, VL1 can bind to a first antigen and VL2 can bind to a second antigen. In some embodiments, VL1 can bind to a first antigen and VL2 can bind to a second antigen. In some embodiments, the bispecific antibody construct comprises both a first polypeptide chain and a second polypeptide chain. The bispecific antibody of this disclosure may be a bivariable domain immunoglobulin (DVD-Ig®) described in Jakob 2013, which combines the target-binding domains of two monoclonal antibodies via a flexible, naturally occurring linker to obtain a tetravalent IgG-like molecule.

[0166] This disclosure further provides a method for producing DVD-Ig binding proteins by pre-selecting parental antibodies against a first antigen and a second antigen. The method for producing a bivariable domain immunoglobulin that binds to two antigens comprises: a) obtaining a first parental antibody or its antigen-binding moiety that binds to a first antigen; b) obtaining a second parental antibody or its antigen-binding moiety that binds to a second antigen; and c) constructing two copies of a first polypeptide chain comprising VH1-(X1)n-VH2-C-(X2)n (wherein VH1 is a first heavy chain variable domain obtained from the first parental antibody or its antigen-binding moiety; VH2 is a second heavy chain variable domain obtained from the second parental antibody or its antigen-binding moiety, which may be the same as or different from the first parental antibody; C is a heavy chain constant domain; (X1)n is a linker, which may or may not be present; and (X2)n is an Fc region). d) the step of constructing two copies of a second polypeptide chain comprising, respectively, VL1-(X1)n-VL2-C-(X2)n (wherein VL1 is a first light chain variable domain obtained from the first parent antibody or its antigen-binding moiety; VL2 is a second light chain variable domain obtained from the second parent antibody or its antigen-binding moiety, which may be the same as or different from the first parent antibody; C is a light chain constant domain; (X1)n is a linker, wherein (X1)n may or may not be present; (X2)n may or may not contain an Fc region; wherein (X2)n may or may not be present); and e) the step of expressing the two copies of the first and second polypeptide chains, such that DVD-Ig that binds to the first and second antigens is produced. Generation of the first antigen-binding and / or second antigen-binding domains

[0167] The variable domain of a DVD-binding protein can be obtained from a parental antibody containing polyclonal and mAbs that bind to the antigen of interest. These antibodies may be naturally occurring, produced by recombinant technology, or designed de novo. mAbs can be prepared using various techniques known in the industry, including the use of hybridoma, recombinant, and phage display technologies, or combinations thereof. Monoclonal antibodies can be prepared by methods disclosed herein.

[0168] A dual variable-domain immunoglobulin (DVD-Ig) molecule is designed so that two different light chain variable domains (VLs), derived from two different parental monoclonal antibodies, followed by a light chain constant domain and, optionally, an Fc region, are linked in series, either directly or via a short linker using recombinant DNA technology. Similarly, the heavy chain comprises two different heavy chain variable domains (VHs) linked in series, followed by a constant domain CH1 and an Fc region. Variable domains can be obtained from parental antibodies produced by any one of the methods described herein using recombinant DNA technology. The variable domains may be mouse heavy chain or light chain variable domains, CDRs, or human heavy chain or light chain variable domains. The first and second variable domains can be linked via a linker sequence, linking them directly to each other using recombinant DNA technology, or the two variable domains can be linked together. The variable domains may bind to the same antigen or to different antigens. The constant domain can be linked to the two linked variable domains using recombinant DNA technology. A sequence containing linked heavy chain variable domains can be linked to a heavy chain constant domain, and a sequence containing linked light chain variable domains can be linked to a light chain constant domain. The constant domains may also be human heavy chain constant domains and human light chain constant domains, respectively. The DVD heavy chain may be further linked to an Fc region. The Fc region may be a natural sequence Fc region, a variant Fc region, or a human Fc region. Two heavy chain DVD polypeptides and two light chain DVD polypeptides can be combined to form a DVD-Ig molecule.

[0169] In some embodiments, the Fc region described herein is derived from an IgG heavy constant chain or an IgA heavy constant chain. In some embodiments, the IgG heavy constant chain includes an IgG1 heavy constant chain. In some embodiments, the IgG1 heavy constant chain includes a human IgG1 heavy constant chain. The amino acid sequence of the wild-type human IgG1 heavy constant chain is provided in Table 23. In some embodiments, the Fc region is engineered not to bind to the Fc gamma receptor (FcγR). In some embodiments, FcγR includes FcγRI, FcγRII, and FcγRIII. [Table 23]

[0170] In some embodiments, the Fc region includes an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the corresponding parent sequence (SEQ ID NO: 199) of the Fc region of IgG1.

[0171] In some embodiments, the Fc region described herein contains one or more mutations compared to the corresponding parent sequence (SEQ ID NO: 199) of the Fc region of IgG1. In some embodiments, the Fc region of IgG1 contains at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, or at least fifteen mutations compared to the corresponding parent sequence of SEQ ID NO: 199. In some embodiments, the Fc region described herein contains at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, or at least fifteen deletions, substitutions, additions, or combinations thereof compared to the corresponding parent sequence of SEQ ID NO: 199. In some embodiments, the Fc region described herein includes at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, or at least fifteen substitutions compared to the corresponding parent sequence of sequence number 199.

[0172] In some embodiments, the Fc region described herein is derived from a human IgG1 heavy chain constant. In some embodiments, the human IgG1 heavy chain constant includes at least one substitution, at least two substitutions, at least three substitutions, at least four substitutions, at least five substitutions, at least six substitutions, or at least seven substitutions. In some embodiments, at least one substitution is selected from EU numbering as N297, C226, C229, E233, L234, L235, G236, G237, P238, F243, M252, S254, T256, D265, S267, H268, D270, P271, R292, Y300, K322, A327, L328, P329, A330, P331, and P396. In some embodiments, at least two substitutions are selected from EU numbering as N297, C226, C229, E233, L234, L235, G236, G237, P238, F243, M252, S254, T256, D265, S267, H268, D270, P271, R292, Y300, K322, A327, L328, P329, A330, P331, and P396. In some embodiments, at least three substitutions are selected from EU numbering as N297, C226, C229, E233, L234, L235, G236, G237, P238, F243, M252, S254, T256, D265, S267, H268, D270, P271, R292, Y300, K322, A327, L328, P329, A330, P331, and P396. In some embodiments, at least four substitutions are selected from EU numbering as N297, C226, C229, E233, L234, L235, G236, G237, P238, F243, M252, S254, T256, D265, S267, H268, D270, P271, R292, Y300, K322, A327, L328, P329, A330, P331, and P396.In some embodiments, at least five substitutions are selected from EU numbering as N297, C226, C229, E233, L234, L235, G236, G237, P238, F243, M252, S254, T256, D265, S267, H268, D270, P271, R292, Y300, K322, A327, L328, P329, A330, P331, and P396. In some embodiments, at least six substitutions are selected from EU numbering as N297, C226, C229, E233, L234, L235, G236, G237, P238, F243, M252, S254, T256, D265, S267, H268, D270, P271, R292, Y300, K322, A327, L328, P329, A330, P331, and P396. In some embodiments, at least seven substitutions are selected from EU numbering as N297, C226, C229, E233, L234, L235, G236, G237, P238, F243, M252, S254, T256, D265, S267, H268, D270, P271, R292, Y300, K322, A327, L328, P329, A330, P331, and P396. In some embodiments, substitutions for N297 include N297A and N297Q substitutions. In some embodiments, substitutions for C226 include C226S substitutions. In some embodiments, substitutions for C229 include C229S substitutions. In some embodiments, substitutions at position E233 include substitutions of E233P and E233D. In some embodiments, substitutions at position L234 include substitutions of L234A, L234D, L234E, L234G, L234H, L234K, L234Q, L234R, L234S, L234T and L234F. In some embodiments, substitutions at position L235 include substitutions of L235A, L235S, L235T, L235H, L235K, L235Q, L235D, L235I, L235V, L235R, L235E and L235G. In some embodiments, substitutions at position G236 include substitutions of G236R.In some embodiments, substitution at position G237 includes substitutions G237A and G237D. In some embodiments, substitution at position P238 includes substitutions P238A, P238D and P238S. In some embodiments, substitution at position F243 includes substitution F243L. In some embodiments, substitution at position M252 includes substitution M252Y. In some embodiments, substitution at position S254 includes substitution S254T. In some embodiments, substitution at position T256 includes substitution T256E. In some embodiments, substitution at position D265 includes substitution D265A. In some embodiments, substitution at position S267 includes substitution S267E. In some embodiments, substitution at position H268 includes substitution H268A and H268D. In some embodiments, substitution at position D270 includes substitution D270A. In some embodiments, substitution at position P271 includes substitution P271G. In some embodiments, substitution at position R292 includes substitution R292P. In some embodiments, substitution at position Y300 includes substitution Y300L. In some embodiments, substitution at position K322 includes substitution K322A and K322Q. In some embodiments, substitution at position A327 includes substitution A327Q and A327G. In some embodiments, substitution at position L328 includes substitution L328E and L328F. In some embodiments, substitution at position P329 includes substitution P329G and P329A. In some embodiments, substitution at position A330 includes substitution A330S, A330R and A330L. In some embodiments, substitution at position P331 includes substitution P331S. In some embodiments, substitution at position P396 includes substitution P396L.

[0173] In some embodiments, the human IgG1 heavy chain constant chain described herein contains two substitutions. In some embodiments, the two substitutions are located at positions L234 and L235 in EU numbering. In some embodiments, the two substitutions are L234A and L235A substitutions.

[0174] In some embodiments, the human IgG1 heavy chain constant chain described herein contains three substitutions. In some embodiments, the three substitutions are located at positions L234, L235, and P329 in EU numbering. In some embodiments, the three substitutions are L234A, L235A, and P329A substitutions. In some embodiments, the three substitutions are located at positions M252, S254, and T256 in EU numbering. In some embodiments, the three substitutions are M252T, S254T, and T256E substitutions.

[0175] In some embodiments, the Fc region includes an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the corresponding parent sequence (SEQ ID NO: 722) of the Fc region of IgG2.

[0176] In some embodiments, the Fc region described herein contains one or more mutations compared to the corresponding parent sequence (SEQ ID NO: 722) of the Fc region of IgG2. In some embodiments, the Fc region of IgG2 contains at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, or at least fifteen mutations compared to the corresponding parent sequence of SEQ ID NO: 722. In some embodiments, the Fc region described herein contains at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, or at least fifteen deletions, substitutions, additions, or combinations thereof compared to the corresponding parent sequence of SEQ ID NO: 722. In some embodiments, the Fc region described herein includes at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, or at least fifteen substitutions compared to the corresponding parent sequence of sequence number 722.

[0177] In some embodiments, the Fc region described herein is derived from a human IgG double chain constant. In some embodiments, the human IgG double chain constant includes at least one substitution, at least two substitutions, at least three substitutions, at least four substitutions, at least five substitutions, at least six substitutions, or at least seven substitutions. In some embodiments, at least one substitution is selected from EU numbering positions C232, C233, V234, G237, P238, M252, S254, T256, H268, N297, V309, A330, and P331. In some embodiments, at least two substitutions are selected from EU numbering C232, C233, V234, G237, P238, M252, S254, T256, H268, N297, V309, A330, and P331. In some embodiments, at least three substitutions are selected from EU numbering C232, C233, V234, G237, P238, M252, S254, T256, H268, N297, V309, A330, and P331. In some embodiments, at least four substitutions are selected from EU numbering C232, C233, V234, G237, P238, M252, S254, T256, H268, N297, V309, A330, and P331. In some embodiments, at least five substitutions are selected from EU numbering C232, C233, V234, G237, P238, M252, S254, T256, H268, N297, V309, A330, and P331. In some embodiments, at least six substitutions are selected from EU numbering C232, C233, V234, G237, P238, M252, S254, T256, H268, N297, V309, A330, and P331. In some embodiments, at least seven substitutions are selected from EU numbering C232, C233, V234, G237, P238, M252, S254, T256, H268, N297, V309, A330, and P331.In some embodiments, substitution at position C232 includes C232S substitution. In some embodiments, substitution at position C233 includes C233S substitution. In some embodiments, substitution at position V234 includes V234A substitution. In some embodiments, substitution at position G237 includes G237A substitution. In some embodiments, substitution at position P238 includes P238S substitution. In some embodiments, substitution at position M252 includes M252Y substitution. In some embodiments, substitution at position S254 includes S254T substitution. In some embodiments, substitution at position T256 includes T256E substitution. In some embodiments, substitution at position H268 includes H268A, H268E, and H268Q substitutions. In some embodiments, substitution at position N297 includes N297A and N297Q substitutions. In some embodiments, substitution at position V309 includes V309L substitution. In some embodiments, the substitution at position A330 includes the substitution A330S. In some embodiments, the substitution at position P331 includes the substitution P331S.

[0178] In some embodiments, the Fc region includes an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the corresponding parent sequence (SEQ ID NO: 723) of the Fc region of IgG4.

[0179] In some embodiments, the Fc region described herein contains one or more mutations compared to the corresponding parent sequence (SEQ ID NO: 723) of the Fc region of IgG4. In some embodiments, the Fc region of IgG4 contains at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, or at least fifteen mutations compared to the corresponding parent sequence of SEQ ID NO: 723. In some embodiments, the Fc region described herein contains at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, or at least fifteen deletions, substitutions, additions, or combinations thereof compared to the corresponding parent sequence of SEQ ID NO: 723. In some embodiments, the Fc region described herein includes at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, or at least fifteen substitutions compared to the corresponding parent sequence of sequence number 723.

[0180] In some embodiments, the Fc region described herein is derived from a human IgG4 heavy chain constant. In some embodiments, the human IgG4 heavy chain constant includes at least one substitution, at least two substitutions, at least three substitutions, at least four substitutions, at least five substitutions, at least six substitutions, or at least seven substitutions. In some embodiments, at least one substitution is selected from EU numbering positions S228, E233, F234, L235, L236, G237, S241, L248, M252, S254, T256, N297, E318, and T394. In some embodiments, at least two substitutions are selected from EU numbering S228, E233, F234, L235, L236, G237, S241, L248, M252, S254, T256, N297, E318, and T394. In some embodiments, at least three substitutions are selected from EU numbering S228, E233, F234, L235, L236, G237, S241, L248, M252, S254, T256, N297, E318, and T394. In some embodiments, at least four substitutions are selected from EU numbering S228, E233, F234, L235, L236, G237, S241, L248, M252, S254, T256, N297, E318, and T394. In some embodiments, at least five substitutions are selected from EU numbering S228, E233, F234, L235, L236, G237, S241, L248, M252, S254, T256, N297, E318, and T394. In some embodiments, at least six substitutions are selected from EU numbering S228, E233, F234, L235, L236, G237, S241, L248, M252, S254, T256, N297, E318, and T394.In some embodiments, at least seven substitutions are selected from EU numbering positions S228, E233, F234, L235, L236, G237, S241, L248, M252, S254, T256, N297, E318, and T394. In some embodiments, the substitution for position S228 includes the S228P substitution. In some embodiments, the substitution for position E233 includes the E233P substitution. In some embodiments, the substitution for position F234 includes the F234V substitution. In some embodiments, the substitution for position L235 includes the L235A substitution. In some embodiments, the substitution for position G237 includes the G237A substitution. In some embodiments, the substitution for position S241 includes the S241P substitution. In some embodiments, the substitution for position L248 includes the L248E substitution. In some embodiments, the substitution at position M252 includes the substitution M252Y. In some embodiments, the substitution at position S254 includes the substitution S254T. In some embodiments, the substitution at position T256 includes the substitution T256E. In some embodiments, the substitution at position N297 includes the substitutions N297A and N297Q. In some embodiments, the substitution at position E318 includes the substitution E318A. In some embodiments, the substitution at position T394 includes the substitution T394D.

[0181] The design of the "bispecific polyvalent full-length binding protein" described herein primarily results in a bivariable domain light chain and a bivariable domain heavy chain that are assembled into a desired "bispecific polyvalent full-length binding protein". Construction of DVD molecules

[0182] A dual variable domain immunoglobulin is disclosed herein. The dual variable domain immunoglobulin (DVD-Ig) molecule is designed so that two different light chain variable domains (VLs), which may be the same or different parental monoclonal antibodies, followed by a light chain constant domain and optionally an Fc region, are linked in series, either directly or by recombinant DNA technology via a short linker. Similarly, the heavy chain comprises two different heavy chain variable domains (VHs) linked in series, followed by a constant domain, a CH1 region and an Fc region.

[0183] Dual variable-domain immunoglobulins containing variable domains are disclosed herein. Variable domains can be obtained from parental antibodies produced by any one of the methods described herein using recombinant DNA technology. In some embodiments, the variable domain is a mouse heavy chain or light chain variable domain. In other embodiments, the variable domain is a CDR-implanted or humanized variable heavy chain or light chain domain. In some embodiments, the variable domain is a human heavy chain or light chain variable domain.

[0184] A dual variable-domain immunoglobulin comprising a first variable domain and a second variable domain is disclosed herein. In one embodiment, the first and second variable domains are directly linked to each other using recombinant DNA technology. In another embodiment, the variable domains are linked via a linker sequence. In one embodiment, two variable domains are linked. Three or more variable domains may also be linked directly or via a linker sequence. The variable domains may bind to the same antigen or to different antigens. The DVD-Ig molecule of the present invention may comprise one immunoglobulin variable domain and one non-immunoglobulin variable domain, such as a receptor ligand-binding domain or an enzyme activity domain. The DVD-Ig molecule may also comprise two or more non-Ig domains.

[0185] In one embodiment, the constant domain is ligated to two ligated variable domains using recombinant DNA technology. In another embodiment, a sequence containing ligated heavy chain variable domains is ligated to a heavy chain constant domain, and a sequence containing ligated light chain variable domains is ligated to a light chain constant domain. In one embodiment, the constant domains are a human heavy chain constant domain and a human light chain constant domain, respectively. The DVD-Ig molecule described herein may also contain a DVD heavy chain. In one embodiment, the DVD heavy chain is further ligated to an Fc region. The Fc region may be a native sequence Fc region or a variant Fc region. In yet another embodiment, the Fc region is human.

[0186] In another embodiment, two heavy-chain DVD polypeptides and two light-chain DVD polypeptides are combined to form a DVD-Ig molecule.

[0187] The binding protein of the present invention can be produced by any of several techniques known in the art. For example, in expression from host cells, an expression vector encoding the DVD heavy chain and DVD light chain is transfected into host cells by standard techniques. Various forms of the term “transfection” are intended to encompass various techniques commonly used for introducing foreign DNA into prokaryotic or eukaryotic host cells, e.g., electroporation, calcium phosphate deposition, DEAE-dextran transfection, etc. While the DVD protein of the present invention can be expressed in prokaryotic or eukaryotic host cells, the DVD protein is expressed in eukaryotic cells, e.g., mammalian host cells, because such eukaryotic cells (particularly mammalian cells) are more likely than prokaryotic cells to fold properly, assemble immunologically active DVD proteins, and secrete them.

[0188] Exemplary mammalian host cells for expressing the recombinant antibody of the present invention include Chinese hamster ovary (CHO cells) (e.g., including dhfr-CHO cells described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selection marker described in Kaufman, RJ and Sharp, PA (1982) Mol. Biol. 159:601-621), NS0 myeloma cells, COS cells, SP2 and PER.C6 cells. When introducing a recombinant expression vector encoding the DVD protein into mammalian host cells, the DVD protein is produced by culturing the host cells for a period sufficient to allow expression of the DVD protein in the host cells or secretion of the DVD protein into the culture medium that promotes host cell growth. The DVD protein can be recovered from the culture medium using standard protein purification methods.

[0189] In the exemplary system for recombinant expression of the DVD protein in the construct described herein, a recombinant expression vector encoding both the DVD heavy chain and the DVD light chain is introduced into dhfr-CHO cells by calcium phosphate-mediated transfection. Within the recombinant expression vector, the DVD heavy chain and light chain genes are operably linked to CMV enhancer / AdMLP promoter regulatory elements, respectively, to drive high levels of gene transcription. The recombinant expression vector also contains the DHFR gene, which allows selection of CHO cells transfected with the vector using methotrexate selection / amplification. Selected transformant host cells are cultured to enable expression of the DVD heavy chain and light chain, and intact DVD protein is recovered from the culture medium. Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect host cells, select transformants, culture host cells, and recover DVD protein from the culture medium. Furthermore, the present invention provides a method for synthesizing the DVD protein of the present invention by culturing the host cells of the present invention in a suitable culture medium until the DVD protein of the present invention is synthesized. The method may further include isolating the DVD protein from the culture medium.

[0190] A key feature of DVD-Ig is that it can be produced and purified in a manner similar to that of conventional antibodies. DVD-Ig production yields a homogeneous, single primary product with desired bispecific activity without any sequence modifications or any kind of chemical alteration of the constant region. Other previously described methods for producing "bispecific," "multispecific," and "multispecific polyvalent" full-length binding proteins do not yield a single primary product, but instead result in the intracellular or secreted production of a mixture of assembled, inactive, monospecific, multispecific, polyvalent, full-length binding proteins and polyvalent full-length binding proteins with different binding site combinations. For example, based on the design described by Miller and Presta (PCT Publication No. WO2001 / 077342(A1)), there are 16 possible combinations of heavy and light chains. As a result, only 6.25% of proteins may be in the desired active form, rather than as a single major product or single primary product, compared to the other 15 possible combinations. Typically, the isolation of the desired, fully active form of protein from inactive and partially active forms of protein using standard chromatographic techniques used in large-scale production has not yet been demonstrated.

[0191] The design of the "bispecific polyvalent full-length binding protein" for use in the constructs described herein primarily results in a bivariable domain light chain and a bivariable domain heavy chain, which are assembled into the desired "bispecific polyvalent full-length binding protein".

[0192] In some embodiments, at least 50%, at least 75%, and at least 90% of the assembled and expressed bivariable domain immunoglobulin molecules are the desired bispecific tetravalent protein. This aspect of the present invention particularly enhances the commercial utility of the present invention. Accordingly, the present invention includes a method for expressing bivariable domain light chains and bivariable domain heavy chains in a single cell, resulting in a single primary product of "bispecific tetravalent full-length binding protein".

[0193] This specification provides a method for expressing bivariable domain light chains and bivariable domain heavy chains in a single cell, wherein the "primary product" yields a "primary product" of "bispecific tetravalent full-length binding protein" that comprises more than 50% of the total assembled protein, including the bivariable domain light chain and bivariable domain heavy chain.

[0194] This specification provides a method for expressing a bivariable domain light chain and a bivariable domain heavy chain in a single cell, wherein the "primary product" yields a single "primary product" of a "bispecific tetravalent full-length binding protein" that comprises more than 75% of all assembled proteins, including the bivariable domain light chain and the bivariable domain heavy chain.

[0195] This specification provides a method for expressing bivariable domain light chains and bivariable domain heavy chains in a single cell, wherein the "primary product" yields a single "primary product" of a "bispecific tetravalent full-length binding protein" that comprises more than 90% of all assembled proteins, including the bivariable domain light chain and bivariable domain heavy chain. binding affinity

[0196] Binding affinity is generally determined by the dissociation constant (K D ) is represented by the K of the antibody. D The value can be determined by any method known in the industry. D Exemplary methods for determining this include using surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), spectroscopic assays, biolayer interferometry (BLI), and grating-coupled interferometry (GCI).

[0197] In some embodiments, the multispecific antibodies described herein include 1 pM to 1 μM, 10 pM to 1 μM, 100 pM to 1 μM, 1 nM to 1 μM, 10 nM to 1 μM, 100 nM to 1 μM, 500 nM to 1 μM, 1 pM to 500 nM, 10 pM to 500 nM, 100 pM to 500 nM, 1 nM to 500 nM, 10 nM to 500 nM, and 100 nM to 500 nM. , including binding affinities in the range of 1 pM to 100 nM, 10 pM to 100 nM, 100 pM to 100 nM, 1 nM to 100 nM, 10 nM to 100 nM, 1 pM to 10 nM, 10 pM to 10 nM, 100 pM to 1 nM, 1 nM to 10 nM, 1 pM to 1 nM, 10 pM to 1 nM, 1 pM to 100 pM, or 10 pM to 100 pM.

[0198] In some embodiments, the BsAb antibodies described herein are 1 pM to 1 μM, 10 pM to 1 μM, 100 pM to 1 μM, 1 nM to 1 μM, 10 nM to 1 μM, 100 nM to 1 μM, 500 nM to 1 μM, 1 pM to 500 nM, 10 pM to 500 nM, 100 pM to 500 nM, 1 nM to 500 nM, 10 nM to 500 nM, 100 nM to 500 nM, 1 pM to 1 Includes average binding affinity to target antigens in the range of 00nM, 10pM-100nM, 100pM-100nM, 1nM-100nM, 10nM-100nM, 1pM-10nM, 10pM-10nM, 100pM-10nM, 1nM-10nM, 1pM-1nM, 10pM-1nM, 100pM-1nM, 1pM-100pM, or 10pM-100pM.

[0199] In some embodiments, the multispecific antibodies described herein include at least two binding domains. In some embodiments, the at least two binding domains are a first binding domain and a second binding domain. In some embodiments, the first binding domain is a TREM1 binding domain. In some embodiments, the second binding domain targets a cytokine of the IL-17 family or IL-17R. In some embodiments, the binding affinity of the multispecific antibody is measured using only one target molecule (e.g., TREM1, sTREM1, a cytokine of the IL-17 family, or IL-17R).

[0200] In some embodiments, the multispecific antibody described herein comprises a TREM1 binding domain and a second binding domain, the second binding domain binding to a cytokine, IL-17R, or a combination thereof derived from the IL-17 family. In some embodiments, the binding affinity of the TREM1 binding domain to TREM1 is lower than the binding affinity of the second binding domain to a cytokine of the IL-17 family or IL-17R. In some embodiments, the multispecific antibody includes a binding affinity of the TREM1 binding domain to TREM1 that is at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 15 times, at least 20 times, at least 40 times, at least 60 times, at least 80(eight) times, or at least 100 times higher than the binding affinity of the second binding domain to a cytokine of the IL-17 family or IL-17R. Alternatively, in some embodiments, the binding affinity of the second binding domain to a cytokine of the IL-17 family or IL-17R is lower than the binding affinity of the TREM1 binding domain to TREM1. Therefore, in some embodiments, the multispecific antibody includes a binding affinity of a second binding domain to IL-17 family cytokines or IL-17R that is at least 2, 3, 4, 5, 10, 15, 20, 40, 60, 80, or at least 100 times higher than the binding affinity of the TREM1-binding domain to TREM1.

[0201] In some embodiments, the multispecific antibody described herein comprises a TREM1 binding domain and a second binding domain, the second binding domain binding to a cytokine, IL-17R, or a combination thereof derived from the IL-17 family. In some embodiments, the binding affinity of the TREM1 binding domain to sTREM1 is lower than the binding affinity of the second binding domain to a cytokine of the IL-17 family or IL-17R. In some embodiments, the multispecific antibody includes a binding affinity of the TREM1 binding domain to sTREM1 that is at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 40, at least 60, at least 80, or at least 100 times higher than the binding affinity of the second binding domain to a cytokine of the IL-17 family or IL-17R. Alternatively, in some embodiments, the binding affinity of the second binding domain to a cytokine of the IL-17 family or IL-17R is lower than the binding affinity of the TREM1 binding domain to sTREM1. Therefore, in some embodiments, the multispecific antibody includes a binding affinity of a second binding domain to IL-17 family cytokines or IL-17R that is at least 2, 3, 4, 5, 10, 15, 20, 40, 60, 80, or at least 100 times higher than the binding affinity of the TREM1 binding domain to sTREM1.

[0202] Alternatively, in some embodiments, the multispecific antibodies described herein undergo a co-binding event. In some embodiments, the multispecific antibody undergoes a positive co-binding event, where the binding of the multispecific antibody to a first target molecule results in an increased binding affinity to a second target molecule. For example, in some embodiments, the binding affinity of a multispecific antibody bound to TREM1 for IL-17 family cytokines or IL-17R is lower than the binding affinity of a multispecific antibody not bound to TREM1 for IL-17 family cytokines or IL-17R. In some embodiments, the binding affinity of a multispecific antibody bound to TREM1 for IL-17 family cytokines or IL-17R is lower than the binding affinity of a multispecific antibody not bound to TREM1 for IL-17 family cytokines or IL-17R. Alternatively, in some embodiments, the multispecific antibody undergoes a negative co-binding event, where the binding of the multispecific antibody to a first target molecule results in a decreased binding affinity to a second target molecule. For example, in some embodiments, the binding affinity of a multispecific antibody bound to TREM1 for IL-17 family cytokines or IL-17R is higher than the binding affinity of a multispecific antibody not bound to TREM1 for IL-17 family cytokines or IL-17R. In some embodiments, the binding affinity of a multispecific antibody bound to TREM1 for IL-17 family cytokines or IL-17R is higher than the binding affinity of a multispecific antibody not bound to TREM1 for IL-17 family cytokines or IL-17R. In some embodiments, the binding affinity of a multispecific antibody bound to sTREM1 for IL-17 family cytokines or IL-17R is lower than the binding affinity of a multispecific antibody not bound to sTREM1 for IL-17 family cytokines or IL-17R. In some embodiments, the binding affinity of a multispecific antibody bound to sTREM1 for IL-17 family cytokines or IL-17R is lower than the binding affinity of a multispecific antibody not bound to sTREM1 for IL-17 family cytokines or IL-17R.Alternatively, in some embodiments, the multispecific antibody undergoes a negative co-binding event, where binding of the multispecific antibody to a first target molecule results in a decrease in binding affinity to a second target molecule. For example, in some embodiments, the binding affinity of a multispecific antibody bound to sTREM1 for IL-17 family cytokines or IL-17R is higher than the binding affinity of a multispecific antibody not bound to sTREM1 for IL-17 family cytokines or IL-17R. Kappa-lambda antibody

[0203] Multispecific (e.g., bispecific, tripspecific) antibodies in the form of kappa-lambda antibodies are provided herein. The bispecific antibodies provided herein have a common heavy chain and two light chains, each having a different specificity—one kappa (K) and one lambda (λ) (i.e., two light chains, two specificities). Methods provided herein produce molecules having specific binding where the diversity is limited to the VL region. These methods produce bispecific antibodies by controlled co-expression and purification of three chains (one VH chain, two VL chains).

[0204] This type of molecule consists of two copies of a unique heavy-chain polypeptide, a first light-chain variable region fused to a constant kappa domain, and a second light-chain variable region fused to a constant lambda domain. Each binding site exhibits different antigen specificity, with contributions from both the heavy and light chains. The light-chain variable regions may be from the lambda or kappa family and are preferably fused to the lambda and kappa constant domains, respectively. This is preferable to avoid the formation of binding sites for non-natural polypeptides. However, the bispecific antibodies of the present invention can also be obtained by fusioning the kappa light-chain variable domain to the constant lambda domain for the first specificity, and the lambda light-chain variable domain to the constant kappa domain for the second specificity.

[0205] An essential step of the exemplary method is the identification of two antibody Fv regions (each composed of a variable light chain and a variable heavy chain domain) having different antigen specificities that share the same heavy chain variable domain. Numerous methods have been described for the generation of monoclonal antibodies and their functional fragments (see, e.g., Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, which is incorporated herein by reference). A fully human antibody is an antibody molecule in which the sequences of both the heavy and light chains, including CDR1 and 2, are derived from human genes. The CDR3 region may be of human origin or may be designed by synthetic means. Such antibodies are referred to herein as "human antibodies" or "fully human antibodies". Human monoclonal antibodies can be prepared by using trioma technology; human B cell hybridoma technology (see Kozbor, et al., 1983 Immunol Today 4: 72); and EBV hybridoma technology for producing human monoclonal antibodies (see Cole, et al, 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies can be produced by using human hybridomas (see Cote, et al, 1983. Proc Natl Acad Sci USA 80: 2026-2030) or by transforming human B cells with Epstein-Barr virus in vitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).

[0206] For example, monoclonal antibodies can be generated by immunizing an animal with a target antigen or an immunogenic fragment, derivative or variant thereof. Alternatively, an animal is immunized with cells transfected with a vector containing a nucleic acid molecule encoding the target antigen such that the target antigen is expressed and associates with the surface of the transfected cells. Various techniques for producing heterologous non-human animals are well known in the art. See, for example, U.S. Patent Nos. 6,075,181 and 6,150,584, which are incorporated herein by reference in their entirety.

[0207] Alternatively, antibodies can be obtained by screening a library containing antibody or antigen-binding domain sequences for binding to the target antigen. This library can be prepared in a bacteriophage (i.e., a "phage display library") as a protein or peptide fusion to a bacteriophage coat protein expressed on the surface of assembled phage particles and on the encoded DNA sequences contained within the phage particles.

[0208] Next, hybridomas obtained from myeloma / B cell fusions are screened for reactivity against the target antigen. For example, monoclonal antibodies can be prepared using the hybridoma method, such as that described by Kohler and Milstein, Nature, 256:495 (1975). In the hybridoma method, a mouse, hamster, or other suitable host animal is typically immunized with an immunizing agent that elicits lymphocytes that produce or are capable of producing antibodies that specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro.

[0209] By using an antibody library in which the heavy chain variable domain is the same for all library members, and thus diversity is limited to the light chain variable domain, it is possible to generate kappa-lambda antibodies having the same heavy chain variable domain. Such libraries are described, for example, in WO2010 / 135558. However, since the light chain variable domain is expressed together with the heavy chain variable domain, both domains may contribute to antigen binding. To further facilitate this process, antibody libraries containing the same heavy chain variable domain and diversity in lambda or kappa variable light chains can be used simultaneously for in vitro selection of antibodies against different antigens. This technique can be used as a building block for generating bispecific antibodies in the fully immunoglobulin form of the present invention, enabling the identification of two antibodies having a common heavy chain, one having a lambda light chain variable domain and the other having a kappa light chain variable domain. Numerous methods for modifying the Fc portion are described and applicable to the antibodies of the present invention (see, for example, Strohl, WR Curr Opin Biotechnol 2009 (6):685-91).

[0210] Another step in the exemplary embodiment is the optimization of the co-expression of a common heavy chain and two different light chains in a single cell to enable the assembly of the bispecific antibody of the present invention. If all polypeptides are expressed at the same level and assemble equally well to form an immunoglobulin molecule, the ratio of single-specificity (same light chain) to bispecificity (two different light chains) should be 50%.

[0211] The simultaneous expression of a heavy chain and two light chains generates a mixture of three different antibodies in the cell culture supernatant: two monospecific bivalent antibodies and one bispecific bivalent antibody. To obtain the molecule of interest, the latter must be purified from the mixture. The method described herein greatly simplifies this purification procedure by using affinity chromatography media that specifically interact with the constant domains of the kappa or lambda light chain, such as CaptureSelect Fab Kappa and CaptureSelect Fab Lambda affinity matrix (BAC BV, Holland). The multi-step affinity chromatography purification method is efficient and generally applicable to the antibodies of the present invention. This is in stark contrast to specific purification methods that need to be developed and optimized for each bispecific antibody derived from a quadroma or other cell line expressing the antibody mixture. Indeed, if the biochemical properties of the different antibodies in the mixture are similar, their separation using standard chromatography techniques such as ion exchange chromatography can be difficult or impossible.

[0212] In some embodiments, the purified bispecific antibodies described herein are characterized as follows. The eluates and eluates from each affinity purification step are analyzed by SDS-PAGE. The specificity and affinity of the κλ isomer are determined by ELISA and surface plasmon resonance. The method of the present invention enables the identification of antibodies at affinities ranging from sub-nanomole to nanomolar without optimization. This is not obvious, as the diversity in the antibody library described herein is limited to light chains that do not contribute much to the binding energy in standard antibodies.

[0213] To avoid the need for access to two antibodies having kappa and lambda-type variable light chain domains, which is considered a limitation of the present invention, the methods described herein enable the production of hybrid light chains in which the lambda variable domain can be fused to the kappa constant domain, and conversely, the kappa variable domain can be fused to the lambda constant domain. In some embodiments, the methods for producing bispecific and / or multispecific antibodies utilize a completely serum-free chemically defined process. These methods include the Chinese hamster ovary (CHO) cell line, the most widely used mammalian cell line in the pharmaceutical industry. Both semi-stable and stable cell lines are produced using the methods described herein. The methods can be used to produce bispecific and / or multispecific antibodies of the present invention on a small scale (e.g., in an Erlenmeyer flask) and a medium scale (e.g., in a 25 L Wave bag). The methods are also readily adaptable to larger-scale production of bispecific and / or multispecific antibodies, as well as antibody mixtures of the present invention. Treatment method

[0214] In some embodiments, methods for treating subjects requiring such treatment are disclosed herein, comprising administering an effective amount of a pharmaceutical composition comprising the multispecific (e.g., bispecific, trispecific) antibody or multispecific (e.g., bispecific, trispecific) molecule described herein or the said multispecific (e.g., bispecific, trispecific) antibody or multispecific (e.g., bispecific, trispecific) molecule described herein to the subject. In some embodiments, the subject may have dementia, frontotemporal dementia, Alzheimer's disease, vascular dementia, mixed dementia, Creutzfeldt-Jakob disease, normal pressure hydrocephalus, amyotrophic lateral sclerosis, Huntington's disease, taupathy, Nasu-Hakola disease, stroke, acute trauma, chronic trauma, cognitive impairment, memory impairment, lupus, acute and chronic colitis, rheumatoid arthritis, atherosclerosis, wound healing, Crohn's disease, inflammatory bowel disease, ulcerative colitis, obesity, essential tremor, central nervous system wolf The subject has one of the following conditions selected from the group consisting of acne, Behçet's disease, Parkinson's disease, Lewy body dementia, multiple system atrophy, Shy-Drager syndrome, progressive supranuclear palsy, corticobasal ganglia degeneration, acute disseminated encephalomyelitis, granulomatous disorders, sarcoidosis, age-related diseases, seizures, spinal cord injury, traumatic brain injury, age-related macular degeneration, glaucoma, retinitis pigmentosa, retinal degeneration, lupus, arthritis, multiple sclerosis, low bone density, osteoporosis, osteogenesis, osteopetrosis, and Paget's disease of bone. In some embodiments, the subject has an autoimmune condition. In some embodiments, autoimmune conditions include inflammatory bowel disease, Crohn's disease, ulcerative colitis, irritable bowel syndrome, rheumatoid arthritis, psoriasis, psoriatic arthritis, systemic lupus erythematosus, lupus nephritis, type 1 diabetes, Graves' disease, multiple sclerosis, autoimmune myocarditis, Kawasaki disease, coronary artery disease, chronic obstructive pulmonary disease, interstitial lung disease, autoimmune thyroiditis, scleroderma, systemic sclerosis, osteoarthritis, atopic dermatitis, vitiligo, graft-versus-host disease, Sjögren's syndrome, autoimmune nephritis, Goodpasture syndrome, chronic inflammatory demyelinating polyneuropathy, allergies, and asthma. In some embodiments, subjects have age-related Alzheimer's disease. In some embodiments, subjects have a disease or condition selected from the group consisting of infectious diseases or autoimmune diseases.In some embodiments, the subject has rheumatoid arthritis, juvenile arthritis, psoriatic arthritis, axial spondyloarthritis, or ankylosing spondylitis. In some embodiments, the subject has psoriasis or sweat gland abscess. In some embodiments, the subject has ulcerative colitis, Crohn's disease, necrotizing enterocolitis, sepsis, or multiple sclerosis. In some embodiments, the subject has sepsis. In some embodiments, the subject has multiple sclerosis.

[0215] Inflammatory diseases or conditions are associated with increased activity and / or expression of TREM-1, IL-17 family cytokines, IL-17R, or combinations thereof in subjects compared to subjects without (or pre-inflammatory) inflammatory diseases or conditions. Furthermore, inflammatory diseases or conditions are also associated with increased activity or expression of one or more downstream inflammatory signaling proteins of TREM1, IL-17 family cytokines, IL-17R, or combinations thereof in subjects compared to subjects without (or pre-inflammatory) inflammatory diseases or conditions. In some embodiments, one or more inflammatory conditions include dementia, frontotemporal dementia, Alzheimer's disease, vascular dementia, mixed dementia, Creutzfeldt-Jakob disease, normal pressure hydrocephalus, amyotrophic lateral sclerosis, Huntington's disease, tauopathic disease, Nasu-Hakola disease, stroke, acute trauma, chronic trauma, cognitive impairment, memory impairment, lupus, acute and chronic colitis, rheumatoid arthritis, atherosclerosis, wound healing, Crohn's disease, inflammatory bowel disease, ulcerative colitis, obesity, and more. This includes essential tremor, lupus of the central nervous system, Behçet's disease, Parkinson's disease, Lewy body dementia, multiple system atrophy, Shy-Drager syndrome, progressive supranuclear palsy, corticobasal ganglia degeneration, acute disseminated encephalomyelitis, granulomatous disorders, sarcoidosis, age-related diseases, seizures, spinal cord injury, traumatic brain injury, age-related macular degeneration, glaucoma, retinitis pigmentosa, retinal degeneration, lupus, arthritis, multiple sclerosis, low bone density, osteoporosis, osteogenesis, osteopetrosis, and Paget's disease of bone. In some embodiments, one or more inflammatory conditions include autoimmune conditions. In some embodiments, one or more inflammatory conditions include inflammatory bowel disease, Crohn's disease, ulcerative colitis, irritable bowel syndrome, rheumatoid arthritis, psoriasis, psoriatic arthritis, systemic lupus erythematosus, lupus nephritis, type 1 diabetes mellitus, Graves' disease, multiple sclerosis, autoimmune myocarditis, Kawasaki disease, coronary artery disease, chronic obstructive pulmonary disease, interstitial lung disease, autoimmune thyroiditis, scleroderma, systemic sclerosis, osteoarthritis, atopic dermatitis, vitiligo, graft-versus-host disease, Sjögren's syndrome, autoimmune nephritis, Goodpasture syndrome, chronic inflammatory demyelinating polyneuropathy, allergies, and asthma. In some embodiments, one or more inflammatory conditions include inflammatory conditions in the central nervous system.In some embodiments, one or more inflammatory conditions include age-related Alzheimer's disease. In some embodiments, one or more inflammatory conditions are selected from the group consisting of infections or autoimmune diseases. In some embodiments, one or more inflammatory conditions include rheumatoid arthritis, juvenile arthritis, psoriatic arthritis, axial spondyloarthritis and / or ankylosing spondylitis. In some embodiments, one or more inflammatory conditions include psoriasis and / or sweat gland abscess. In some embodiments, one or more inflammatory conditions include ulcerative colitis, Crohn's disease, necrotizing enterocolitis, sepsis and / or multiple sclerosis. In some embodiments, one or more inflammatory conditions include sepsis and / or multiple sclerosis. Accordingly, in some embodiments, a method for treating an inflammatory disease or condition in a subject includes administering a BsAb described herein, the BsAb reducing the activity and / or expression of TREM1, IL-17 family cytokines, IL-17R, one or more of their downstream signaling proteins, or combinations thereof in a subject compared to a subject treated with a monospecific antibody targeting TREM1, IL-17 family cytokines, or IL-17R.

[0216] In some embodiments, a multispecific (e.g., bispecific, triplicate) antibody or multispecific (e.g., bispecific, triplicate) molecule is administered together with one or more additional therapeutic agents. In some embodiments, a multispecific (e.g., bispecific, triplicate) antibody or multispecific (e.g., bispecific, triplicate) molecule and one or more additional therapeutic agents are administered simultaneously. In some embodiments, a multispecific (e.g., bispecific, triplicate) antibody or multispecific (e.g., bispecific, triplicate) molecule and one or more additional therapeutic agents are administered sequentially. Diagnostic methods

[0217] In some embodiments, a method for diagnosing the condition of a subject is disclosed herein, comprising incubating a sample with a composition comprising an effective amount of a multispecific (e.g., bispecific, triplicate) antibody or multispecific (e.g., bispecific, triplicate) molecule as described herein. In some embodiments, the sample includes tissue, blood, serum, plasma, saliva, urine, or a combination thereof. In some embodiments, the diagnosis is based on the expression level of TREM1, where an increase in the level of TREM1 in the subject compared to the level of TREM1 in a healthy individual indicates that the individual is suffering from an autoimmune condition. In some embodiments, the diagnosis is based on the expression level of TREM1, where an increase in the level of TREM1 in the subject compared to the level of TREM1 in the subject before the onset of the autoimmune condition. In some embodiments, the diagnosis is based on the expression level of sTREM1, where an increase in the level of sTREM1 in the subject compared to the level of sTREM1 in a healthy individual indicates that the individual is suffering from an autoimmune condition. In some embodiments, the diagnosis is based on the expression level of sTREM1, where the level of sTREM1 in the subject is elevated compared to the level of sTREM1 in the subject prior to the onset of the autoimmune condition. In some embodiments, the autoimmune condition includes inflammatory bowel disease, Crohn's disease, ulcerative colitis, irritable bowel syndrome, rheumatoid arthritis, psoriasis, psoriatic arthritis, systemic lupus erythematosus, lupus nephritis, type 1 diabetes mellitus, Graves' disease, multiple sclerosis, autoimmune myocarditis, Kawasaki disease, coronary artery disease, chronic obstructive pulmonary disease, interstitial lung disease, autoimmune thyroiditis, scleroderma, systemic sclerosis, osteoarthritis, atopic dermatitis, vitiligo, graft-versus-host disease, Sjögren's syndrome, autoimmune nephritis, Goodpasture syndrome, chronic inflammatory demyelinating polyneuropathy, allergies, and asthma. In some embodiments, the autoimmune condition includes rheumatoid arthritis, juvenile arthritis, psoriatic arthritis, axial spondyloarthritis, ankylosing spondylitis, psoriasis, sweat gland abscess, ulcerative colitis, Crohn's disease, necrotizing enterocolitis, sepsis, multiple sclerosis, or a combination thereof. In some embodiments, the autoimmune condition includes sepsis. In some embodiments, the autoimmune condition includes multiple sclerosis. Dosage

[0218] Compositions comprising multispecific (e.g., bispecific, tripspecific) antibodies or multispecific (e.g., bispecific, tripspecific) molecules or their antigen-binding fragments for the treatment (including prevention) of diseases (e.g., infectious conditions, disorders or diseases, autoimmune conditions, disorders or diseases, skin conditions, disorders or diseases). In some embodiments, the composition is a pharmaceutical composition comprising a pharmaceutically acceptable carrier. The composition is administered in an amount effective for the treatment (including prevention) of infectious conditions, disorders or diseases, autoimmune conditions, disorders or diseases, skin conditions, disorders or diseases. In some embodiments, the composition (e.g., an antibody or its antigen-binding fragment or a nucleic acid molecule encoding the antibody or its antigen-binding fragment) is administered in an amount effective for enhancing the immune response and / or increasing T cell activation in a subject. The composition should be used for in vivo administration to a subject by any available means, such as parenteral administration. For administration to a subject, the compositions or pharmaceuticals comprising antibodies or their antigen-binding fragments described herein may be sterile, which can be readily achieved by filtration through a sterile filtration membrane or by other methods known to those skilled in the art. In one embodiment, the composition or pharmaceutical is treated to be free of pyrogens or endotoxins. Testing pharmaceutical compositions or pharmaceuticals for pyrogens or endotoxins, and preparing pharmaceutical compositions or pharmaceuticals free of pyrogens or endotoxins, or preparing pharmaceutical compositions or pharmaceuticals containing endotoxins at clinically acceptable levels, is well understood by those skilled in the art. Commercially available kits are available for testing pharmaceutical compositions or pharmaceuticals for pyrogens or endotoxins.

[0219] The compositions used for in vivo administration, such as parenteral administration, in the methods described herein may be sterile, which can be readily achieved by filtration through a sterile filtration membrane or by other methods known to those skilled in the art.

[0220] In some embodiments, elevated levels of TREM1 and / or sTREM1 directly correlate with the condition. In some embodiments, the condition includes rheumatoid arthritis, juvenile arthritis, psoriatic arthritis, axial spondyloarthritis, ankylosing spondylitis, psoriasis, sweat gland abscess, ulcerative colitis, Crohn's disease, necrotizing enterocolitis, sepsis, multiple sclerosis, or a combination thereof. In some embodiments, the condition includes sepsis. In some embodiments, the condition includes multiple sclerosis. In some embodiments, the dose for treating the condition in a subject is determined based on its proportional increase compared to TREM1 and / or sTREM1 in a healthy individual. In some embodiments, the dose for treating the condition in a subject is determined based on a proportional change in TREM1 and / or sTREM1 levels compared to the levels of TREM1 and / or sTREM1 before the onset of the condition. Pharmaceutical compositions and dosage forms

[0221] In certain embodiments, pharmaceutical compositions comprising multispecific (e.g., bispecific, triplicate) antibodies or multispecific (e.g., bispecific, triplicate) molecules or functional fragments thereof for administration to a target are disclosed herein.

[0222] In some embodiments, pharmaceutical compositions comprising multispecific (e.g., bispecific, trispecific) molecules or multispecific (e.g., bispecific, trispecific) antibodies described herein are formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and adjuvants that facilitate the processing of the active compound into a pharmaceutically usable preparation. The appropriate formulation depends on the selected route of administration. Outlines of the pharmaceutical compositions described herein can be found, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, HA and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999).

[0223] Pharmaceutical compositions are manufactured in conventional ways, as needed, including, but not limited to, conventional mixing, dissolution, granulation, sugar-coated tablet preparation, powdering, emulsification, encapsulation, capture, or compression processes.

[0224] In certain embodiments, the composition may also contain one or more pH adjusters or buffering agents, including acids such as acetic acid, boric acid, citric acid, lactic acid, phosphoric acid, and hydrochloric acid; bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate, and tris-hydroxymethylaminomethane; and buffers such as citrate / dextrose, sodium bicarbonate, and ammonium chloride. Such acids, bases, and buffers are included in amounts necessary to maintain the pH of the composition within an acceptable range.

[0225] In other embodiments, the composition may also contain one or more salts in amounts necessary to bring the osmolality of the composition into an acceptable range. Such salts include those having sodium, potassium or ammonium cations and chlorides, citric acid, ascorbic acid, boric acid, phosphoric acid, bicarbonate, sulfuric acid, thiosulfate or bisulfite anions; preferred salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite and ammonium sulfate.

[0226] The pharmaceutical compositions described herein are administered by any preferred route of administration, including, but not limited to, oral, parenteral (e.g., intravenous, subcutaneous, intramuscular, intracerebral, intraventricular, intraarticular, intraperitoneal, intraarterial, intrathecal, lymphatic, intralesional, intraarticular, intracapsular, intraorbital, intracardiac, intradermal, transdermal, subepidermal, subcapsular, subarachnoid, intraspinal, epidural, intrasternal, or intracranial), intranasal, buccal, sublingual, spinal, or rectal administration routes. In some embodiments, the pharmaceutical compositions are formulated for parenteral administration (e.g., intravenous, subcutaneous, intramuscular, intracerebral, intraventricular, intraarticular, intraperitoneal, intraarterial, intrathecal, lymphatic, intralesional, intraarticular, intraarticular, intraarticular, intraarticular, intraarticular, intraarticular, intraarticular, intraarticular, intraarticular, intraarticular, intraarticular, intraorbital, intraorbital, intracardiac, intradermal, transdermal, subepidermal, subcapsular, subarachnoid, intraspinal, epidural, intrasternal, or intracranial).

[0227] The pharmaceutical compositions described herein can be formulated into any suitable dosage form, including, but not limited to, aqueous oral dispersions, liquids, gels, syrups, elixirs, slurries, suspensions, etc. for oral ingestion by the subject being treated, solid oral dosage forms, aerosols, controlled release formulations, fast-dissolving formulations, effervescent formulations, lyophilized formulations, tablets, powders, pills, dragees, capsules, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations, and mixed immediate release and controlled release formulations.

[0228] In some embodiments, the pharmaceutical compositions described herein include one or more stabilizers, fillers, surfactants, or combinations thereof. In some embodiments, the stabilizers include amino acids (e.g., glycine, alanine, lysine, arginine, and threonine), carbohydrates (e.g., glucose, sucrose, trehalose, raffinose, and maltose), polyols (e.g., glycerol, mannitol, sorbitol, cyclodextrin or dextran of any type and molecular weight, and polyethylene glycol (PEG)). In some embodiments, the fillers include lactose, sucrose, glucose, mannitol, sorbitol, calcium carbonate, and magnesium stearate. In some embodiments, the surfactants include anionic surfactants, cationic surfactants, zwitterionic surfactants, or nonionic surfactants. In some embodiments, the surfactants include alkyl ethoxylates, nonylphenol ethoxylates, amine ethoxylates, polyethylene oxide, polypropylene oxide, fatty alcohols (e.g., cetyl alcohol or oleyl alcohol), cocamide MEA, cocamide DEA, polysorbates, and dodecyldimethylamine oxide. In some embodiments, the surfactant is polysorbate 20. In some embodiments, the surfactant is polysorbate 80.

[0229] In some embodiments, the pharmaceutical compositions described herein are formulated into capsules. In some embodiments, the pharmaceutical compositions are formulated into solutions (e.g., for intravenous administration). In some embodiments, the pharmaceutical compositions are formulated as infusions. In some embodiments, the pharmaceutical compositions are formulated as injections.

[0230] The pharmaceutical solid dosage forms described herein may optionally include the compounds described herein and one or more pharmaceutically acceptable additives, such as compatible carriers, binders, fillers, suspending agents, flavoring agents, sweeteners, disintegrants, dispersants, surfactants, lubricants, colorants, diluents, solubilizers, humectants, plasticizers, stabilizers, penetration enhancers, wetting agents, defoamers, antioxidants, preservatives, or one or more combinations thereof.

[0231] In further embodiments, a film coating is provided around the composition using a standard coating procedure, such as that described in Remington's Pharmaceutical Sciences, 20th Edition (2000). In some embodiments, the composition is formulated into particles (e.g., for administration by capsule), and some or all of the particles are coated. In some embodiments, the composition is formulated into particles (e.g., for administration by capsule), and some or all of the particles are microencapsulated. In some embodiments, the composition is formulated into particles (e.g., for administration by capsule), and some or all of the particles are not microencapsulated and are not coated.

[0232] In certain embodiments, the compositions provided herein may include one or more preservatives for inhibiting microbial activity. Suitable preservatives include mercury-containing substances such as merfen and thiomersal; stabilized chlorine dioxide; and quaternary ammonium compounds such as benzalkonium chloride, cetyltrimethylammonium bromide, and cetylpyridinium chloride.

[0233] The compositions disclosed herein, comprising the antibody or antigen-binding fragment described herein, may also contain more than one active compound required for the specific indication being treated, preferably having complementary activities that do not adversely affect each other. For example, the compositions may contain retinoids such as acitretin (e.g., Soriatane®) and isotretinoin, immunosuppressants (e.g., rapamycin, T-cell blockers [e.g., Amevive® (Alefacept) and Raptiva® (Ephalizumab)], cyclosporine, methotrexate, mycophenolate mofetil, mycophenolic acid, leflunomide, tacrolimus, etc.), hydroxyureas (e.g., Hydro ea (registered trademark), sulfasalazine, 6-thioguanine, fumarate (e.g., dimethyl fumarate and fumarate esters), azathioprine, colchicine, alitretinoin, steroids, corticosteroids, certolizumab, aprelimast, mometasone, rosiglitazone, pioglitazone, botulinum toxin, triamcinolone, IFN-1 (InflaRx), bimekizumab (UCB), MaBpl (XBiotech), LY-3041658 (Eli Lilly), TE-2232 (Immunwork), NSAIDs, prescription anesthetics, ketoprofen, codeine, gabapentin, pregabalin gentanil, antibiotics (topic, oral, IV) (e.g., clindamycin, rifampin, tetracycline, thalecycline, doxycycline, minocycline, rimecycline, trimethoprim-sulfamethoxazole, erythromycin, ceftriaxone, moxifloxacin, metronidazole) The following may further be included: corticosteroids (injection or oral), antiandrogen / hormone therapy (oral contraceptives, spironolactone, finasteride, dutasteride, progesterone IUD, cyproterone acetate, ethiniroestradiol, gestodene, norgestimate, desogestrel, drospirenone, spironolactone), triamcinolone acetonide, MEDI8968, hydroxychloroquine, dapsone, metformin, adapalene, azelaic acid, and zinc.Such molecules are preferably present in combination in amounts effective for the intended purpose. The active components of compositions comprising the antibodies or antigen-binding fragments described herein can also be captured, for example, in microcapsules prepared by droplet formation techniques or interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microparticles, microemulsions, nanoparticles and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences (16th ed., Osol, ed., 1980). Pharmaceutical compositions can also be delivered in vesicles, particularly liposomes (see Langer 1990 Science 249:1527-1533; Treat et al. (1989) in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365; Lopez-Berestein, ibid., pp. 317-327; see the same book in general). Liposomes, including emulsions, foams, micelles, insoluble monolayers, phospholipid dispersions, and lamellar layers, can serve as vehicles for targeting M-CSF antibodies to specific tissues and for increasing the half-life of compositions. For example, various methods for preparing liposomes are available, as described in U.S. Patent Nos. 4,837,028 and 5,019,369 (these patents are incorporated herein by reference).

[0234] For oral, buccal, and sublingual administration, powders, suspensions, granules, tablets, pills, capsules, gel caps, and caplets are acceptable solid dosage forms. These can be prepared, for example, by mixing one or more compounds of the present invention, or pharmaceutically acceptable salts or tautomers thereof, with at least one additive, such as starch or other additives. Suitable additives include sucrose, lactose, cellulose sugars, mannitol, maltitol, dextran, starch, agar, alginate, chitin, chitosan, pectin, tragacanth gum, acacia gum, gelatin, collagen, casein, albumin, synthetic or semi-synthetic polymers, or glycerides. If necessary, the oral dosage form may contain other components to aid administration, such as inert diluents, lubricants such as magnesium stearate, preservatives such as parabens or sorbic acid, antioxidants such as ascorbic acid, tocopherol or cysteine, disintegrants, binders, thickeners, buffers, sweeteners, flavorings or fragrances. The tablets and pills may be further treated with suitable coating materials known in the industry.

[0235] Liquid dosage forms for oral administration may be in the form of pharmaceutically acceptable emulsions, syrups, elixirs, suspensions, and solutions, which may contain an inert diluent such as water. In some embodiments, pharmaceutical formulations and pharmaceuticals may be prepared as liquid suspensions or aqueous solutions using sterile liquids, such as, but not limited to, oils, water, alcohols, and combinations thereof. In some embodiments, pharmaceutical compositions may be prepared in lyophilized form. Lyophilized preparations may contain cryoprotectants known in the art. As used herein, the term “cryoprotectant” generally includes agents that provide stability to proteins from stress induced by freezing. Examples of cryoprotectants include, for example, polyols such as mannitol, sugars such as sucrose, and surfactants such as polysorbates, poloxamers, or polyethylene glycol. Cryoprotectants also contribute to the isotonicity of the formulation. Pharmaceutically suitable surfactants, suspensions, and emulsifiers may be added for oral or parenteral administration.

[0236] Injectable dosage forms generally include aqueous or oily suspensions that can be prepared using suitable dispersants or wetting agents and suspending agents. The injectable form may be in the liquid phase or in the form of a suspension, prepared with a solvent or diluent. Acceptable solvents or vehicles include sterile water, Ringer's solution, or isotonic aqueous saline solutions. Alternatively, sterile oil may be used as a solvent or suspending agent. Preferably, the oil or fatty acid is non-volatile and includes natural or synthetic oils, fatty acids, mono, di, or triglycerides.

[0237] For injection, the pharmaceutical formulation and / or pharmaceutical may be a powder suitable for reconstitution with the appropriate solutions described above. Examples of these include, but are not limited to, lyophilized, tumble-dried or spray-dried powders, amorphous powders, granules, precipitates, or particles. For injection, the formulation may optionally contain stabilizers, pH modifiers, surfactants, bioavailability modifiers, and combinations thereof.

[0238] In some embodiments, the pharmaceutical compositions described herein include (a) one or more antibodies described herein; and (b) at least one buffer, stabilizer, pH modifier, salt, and surfactant. kit

[0239] Kits, drugs, compositions, and unit dosage forms for use in any of the methods described herein are also provided herein. Kits comprising a therapeutically effective amount of at least one multispecific (e.g., bispecific, triplicate) antibody or multispecific (e.g., bispecific, triplicate) molecule or antigen-binding fragment thereof as disclosed herein are provided herein. In some embodiments, the kit further comprises a second therapeutic agent (e.g., an immunosuppressant, an immunomodulator, or other agent including, but not limited to, agents disclosed herein). In some embodiments, the antibody or antigen-binding fragment thereof is in aqueous or lyophilized form. The kit further comprises a diluent or reconstitution solution.

[0240] The kit may include one or more containers containing the antibody (or unit dosage form and / or product). In some embodiments, a unit dose is provided that contains a predetermined amount of an antibody-containing composition (e.g., a therapeutically effective dose), with or without one or more further agents. In some embodiments, such a unit dose is supplied in a single-use, pre-filled syringe for injection. In some embodiments, the composition containing the antibody or its antigen-binding fragment may contain buffers such as saline, sucrose, etc.; phosphates, etc.; and / or be formulated within a stable and effective pH range. In some embodiments, the antibody or its antigen-binding fragment may be provided as a lyophilized powder that can be restored upon addition of a suitable liquid, e.g., sterile water. In some embodiments, the antibody or its antigen-binding fragment further comprises, but is not limited to, one or more substances that inhibit protein aggregation, such as sucrose and arginine. In some embodiments, the antibody or its antigen-binding fragment further comprises heparin and / or proteoglycans.

[0241] In some embodiments, the kit further includes instructions for use in the treatment of a disease or condition according to any of the methods described herein. The kit may further include instructions for individual preferred treatment selections. Instructions for use supplied with the kit are typically written instructions on a label or accompanying documents (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable. In some embodiments, the kit further includes another therapeutic agent.

[0242] The kit is contained in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, wide-mouthed bottles, and flexible packaging (e.g., sealed Mylar bags or plastic bags). The kit may optionally provide additional components such as cushioning and interpretive information. Thus, this application also provides products including vials (such as sealed vials), bottles, wide-mouthed bottles, and flexible packaging. Exemplary Embodiments

[0243] Disclosed herein are bispecific molecules comprising a first domain and a second domain, wherein the first domain binds to TREM1 or a functional fragment thereof, and the second domain binds to at least one member of the IL-17 family of interleukins, the IL-17 receptor (IL-17R), or a functional fragment or combination thereof. Figure 1 depicts an exemplary bispecific antibody comprising a TREM1-binding domain and an IL-17-binding domain, wherein the IL-17-binding domain can bind to a cytokine of the IL-17 family, IL-17R, or a combination thereof. In some embodiments, the bispecific molecule is an antibody, a variant of an antibody, or an engineered functional fragment of an antibody. In some embodiments, the interleukin comprises one of IL17A, IL17A / F, or a combination thereof or a functional fragment. In some embodiments, TREM1 comprises an IgV domain or a stem region. In some embodiments, the bispecific molecule is a Fab2 antibody, a bis-scFv antibody, a diabody, DVD-Ig, TandAb, tandem scFv-Fc, one-arm tandem scFv-Fc, DART, DART-Fc, or a functional fragment thereof. In some embodiments, the interleukin is IL-17 or a functional fragment thereof. In some embodiments, the bispecific molecule includes a heterodimer antibody or a functional fragment thereof. In some embodiments, the bispecific molecule includes a constant region. In some embodiments, the molecule includes a sequence knockout in the constant region. In some embodiments, the first domain includes a TREM1-binding heavy chain variable domain. In some embodiments, the first domain includes a TREM1-binding light chain variable domain. In some embodiments, the second domain includes an interleukin-binding heavy chain variable domain. In some embodiments, the second domain includes an interleukin-binding light chain variable domain. In some embodiments, at least one of the first and second domains includes a light chain constant domain and / or a heavy chain constant domain.

[0244] Also disclosed herein are bispecific molecules for use in the treatment of diseases or conditions. In some embodiments, the disease or condition is selected from the group consisting of rheumatoid arthritis, juvenile arthritis, psoriatic arthritis, axial spondyloarthritis, or ankylosing spondylitis. In some embodiments, the bispecific molecule is for use in the treatment of psoriasis or sweat gland abscesses. In some embodiments, the bispecific molecule is for use in the treatment of ulcerative colitis, Crohn's disease, necrotizing enterocolitis, sepsis, or multiple sclerosis. In some embodiments, the bispecific molecule comprises at least one of the Fc region and / or Fab region.

[0245] Also disclosed herein are pharmaceutical compositions comprising the bispecific molecules described herein and pharmaceutically acceptable carriers. Also disclosed herein are methods for treating a disease or condition in a subject, comprising administering an effective amount of the bispecific molecule or pharmaceutical composition described in any one of the claims to the subject, thereby treating the disease or condition.

[0246] Also disclosed herein are methods for treating infectious diseases, autoimmune diseases and / or skin diseases in a subject, comprising administering an effective amount of a bispecific molecule to the subject, comprising a TREM1-binding domain or its TREM1-binding variant, which comprises at least one of the heavy chain complementarity-determining regions (CDR-H) described in Table 8 or the light chain CDR-L described in Table 10; and an interleukin or its TREM1-binding variant, which comprises at least one of the heavy chain complementarity-determining regions (CDR-H) described in Table 2 or the light chain CDR-L described in Table 4.

[0247] Also disclosed herein are compositions comprising a bispecific molecule comprising a first domain and a second domain, wherein the first domain binds to TREM1 or a functional fragment thereof, and the second domain binds to an interleukin or a functional fragment thereof, and administration of an effective amount of the composition to a subject requiring it results in treatment of a disease or condition. In some embodiments, the interleukin comprises IL-17A, IL-17A / F, or any combination thereof. In some embodiments, the bispecific molecule is a Fab2 antibody, a bis-scFv antibody, a Diabody, DVD-Ig, TandAb, a tandem scFv-Fc, a one-arm tandem scFv-Fc, DART, DART-Fc, or a functional fragment thereof. In some embodiments, the interleukin is IL-17 or a functional fragment thereof. In some embodiments, the bispecific molecule comprises a heterodimer antibody or a functional fragment thereof. In some embodiments, the bispecific molecule comprises a constant region. In some embodiments, the bispecific molecule comprises a sequence knockout in the constant region. Furthermore, nucleic acids encoding at least a portion of any one of the bispecific molecules disclosed herein are also disclosed herein. Further exemplary embodiments

[0248] Embodiment 1: A bispecific molecule comprising a first domain and a second domain, wherein the first domain binds to TREM1 or a functional fragment thereof, and the second domain binds to IL-17, IL-17R, or a functional fragment thereof.

[0249] Embodiment 2: The bispecific molecule according to Embodiment 1, which is an antibody, an antibody variant, or an engineered functional fragment of an antibody.

[0250] Embodiment 3: The bispecific molecule according to Embodiment 1 or 2, wherein IL-17 comprises one of IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, IL-17F, IL-17A / F, or a combination thereof or a functional fragment.

[0251] Embodiment 4: A bispecific molecule according to any one of Embodiments 1 to 3, wherein the binding affinity of the first domain to TREM1 is lower than the binding affinity of the second domain to IL-17, IL-17R, or a functional fragment thereof.

[0252] Embodiment 5: The bispecific molecule according to Embodiment 4, wherein the binding affinity of the second binding domain to IL-17, IL-17R, or a functional fragment thereof is at least twice the binding affinity of the first domain to TREM1, and is lower than the binding affinity of the second domain to IL-17, IL-17R, or a functional fragment thereof.

[0253] Embodiment 6: A bispecific molecule according to any one of Embodiments 1 to 3, wherein the binding affinity of the first domain to TREM1 is higher than the binding affinity of the second domain to IL-17, IL-17R, or a functional fragment thereof.

[0254] Embodiment 7: The bispecific molecule according to Embodiment 6, wherein the binding affinity of the first domain to TREM1 is at least twice that of the binding affinity of the second binding domain to IL-17, IL-17R, or a functional fragment thereof.

[0255] Embodiment 8: The bispecific molecule according to any one of Embodiments 1 to 7, wherein the bispecific molecule is Fab2 antibody, bis-scFv antibody, diabody, DVD-Ig, TandAb, tandem scFv-Fc, 1-arm tandem scFv-Fc, DART, DART-Fc, or a functional fragment thereof.

[0256] Embodiment 9: A bispecific molecule according to any one of Embodiments 1 to 8, wherein the first domain comprises a TREM1-binding heavy chain variable domain.

[0257] Embodiment 10: A bispecific molecule according to any one of Embodiments 1 to 9, wherein the first domain comprises a TREM1-binding light chain variable domain.

[0258] Embodiment 11: A bispecific molecule according to any one of Embodiments 1 to 10, wherein the second domain comprises an IL-17 binding heavy chain variable domain.

[0259] Embodiment 12: A bispecific molecule according to any one of Embodiments 1 to 11, wherein the second domain comprises an IL-17 binding light chain variable domain.

[0260] Embodiment 13: A bispecific molecule according to any one of Embodiments 1 to 12, wherein at least one of the first domain and the second domain comprises a light chain constant domain and / or a heavy chain constant domain.

[0261] Embodiment 14: The bispecific molecule according to any one of Embodiments 1 to 13, wherein the bispecific molecule comprises at least one of the Fc region and / or the Fab region.

[0262] Embodiment 15: The bispecific molecule according to Embodiment 14, wherein the Fc region comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to any one of the amino acid sequences of SEQ ID NOs. 199, 722, and 723.

[0263] Embodiment 16: The bispecific molecule according to Embodiment 14, wherein the heavy chain constant domain of the first domain comprises an Fc region having S354C mutations and T366W mutations in EU numbering, and the heavy chain constant domain of the second domain comprises an Fc region having Y349C mutations, T366S mutations and Y407V mutations in EU numbering.

[0264] Embodiment 17: The bispecific molecule according to Embodiment 14, wherein the heavy chain constant domain of the second domain comprises an Fc region having S354C mutations and T366W mutations in EU numbering, and the heavy chain constant domain of the first domain comprises an Fc region having Y349C mutations, T366S mutations and Y407V mutations in EU numbering.

[0265] Embodiment 18: A bispecific molecule according to Embodiment 14, wherein the Fc region comprises a human IgG1 heavy chain constant chain having at least one substitution selected from the EU numbering positions N297, C226, C229, E233, L234, L235, G236, G237, P238, F243, M252, S254, T256, D265, S267, H268, D270, P271, R292, Y300, K322, A327, L328, P329, A330, P331, and P396.

[0266] Embodiment 19: The bispecific molecule according to Embodiment 14, wherein the Fc region comprises a human IgG double-chain constant chain having at least one substitution selected from EU numbering positions C232, C233, V234, G237, P238, M252, S254, T256, H268, N297, V309, A330, and P331.

[0267] Embodiment 20: The bispecific molecule according to Embodiment 14, wherein the Fc region comprises a human IgG4 heavy chain constant chain having at least one substitution selected from EU numbering positions S228, E233, F234, L235, L236, G237, S241, L248, M252, S254, T256, N297, E318, and T394.

[0268] Embodiment 21: A bispecific molecule according to any one of Embodiments 1 to 20, comprising at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, or higher anti-inflammatory activity compared to a combination of a monospecific antibody that binds to TREM1 and a monospecific antibody that binds to a cytokine of the IL-17 family, IL-17R, or a combination thereof.

[0269] Embodiment 22: A bispecific molecule according to any one of Embodiments 1 to 21, comprising pH-dependent target binding activity.

[0270] Embodiment 23: A bispecific molecule according to any one of Embodiments 1 to 22, comprising neuroprotective activity.

[0271] Embodiment 24: A bispecific molecule according to any one of Embodiments 1 to 23 for use in the treatment of a disease or condition.

[0272] Embodiment 25: The bispecific molecule according to Embodiment 24, wherein the disease or condition is selected from the group consisting of rheumatoid arthritis, juvenile arthritis, psoriatic arthritis, axial spondyloarthritis, or ankylosing spondylitis.

[0273] Embodiment 26: A bispecific molecule according to any one of Embodiments 1 to 23 for use in the treatment of psoriasis or sweat gland abscesses.

[0274] Embodiment 27: A bispecific molecule according to any one of Embodiments 1 to 23 for use in the treatment of ulcerative colitis, Crohn's disease, necrotizing enterocolitis, sepsis, or multiple sclerosis.

[0275] Embodiment 28: A bispecific molecule according to any one of Embodiments 1 to 23 for use in the treatment of sepsis.

[0276] Embodiment 29: A bispecific molecule according to any one of Embodiments 1 to 23 for use in the treatment of multiple sclerosis.

[0277] Embodiment 30: A bispecific molecule according to any one of Embodiments 1 to 23 for use in the treatment of age-related Alzheimer's disease.

[0278] Embodiment 31: A bispecific molecule comprising a first heavy chain, a second heavy chain, and a light chain as described in any one of the candidates provided in Table 24.

[0279] Embodiment 32: A composition comprising the bispecific molecule described in any one of Embodiments 1 to 31.

[0280] Embodiment 33: A composition comprising a bispecific molecule having a first domain and a second domain, wherein the first domain binds to TREM1 or a functional fragment thereof, and the second domain binds to IL-17, IL-17R or a functional fragment thereof, wherein administration of an effective amount of the composition to a subject requiring it results in treatment of a disease or condition.

[0281] Embodiment 34: The composition according to Embodiment 33, wherein IL-17 comprises IL-17A, IL-17A / F, or any combination thereof.

[0282] Embodiment 35: The composition according to Embodiment 33 or 34, wherein the disease or condition is selected from the group consisting of rheumatoid arthritis, juvenile arthritis, psoriatic arthritis, ankylosing spondylitis, axial spondyloarthritis, psoriasis, sweat gland abscess, ulcerative colitis, Crohn's disease, necrotizing enterocolitis, sepsis, or multiple sclerosis.

[0283] Embodiment 36: The composition according to Embodiment 33 or 34, wherein the disease or condition is rheumatoid arthritis, juvenile arthritis, psoriatic arthritis, axial spondyloarthritis, or ankylosing spondylitis.

[0284] Embodiment 37: The composition according to Embodiment 33 or 34, wherein the disease or condition is psoriasis or sweat gland abscess.

[0285] Embodiment 38: The composition according to Embodiment 33 or 34, wherein the disease or condition is ulcerative colitis, Crohn's disease, necrotizing enterocolitis, sepsis, or multiple sclerosis.

[0286] Embodiment 39: The composition according to Embodiment 33 or 34, wherein the disease or condition is sepsis.

[0287] Embodiment 40: The composition according to Embodiment 33 or 34, wherein the disease or condition is multiple sclerosis.

[0288] Embodiment 41: The composition according to Embodiment 33 or 34, wherein the disease or condition is age-related Alzheimer's disease.

[0289] Embodiment 42: A nucleic acid encoding at least a portion of the bispecific molecule described in any one of Embodiments 1 to 31 or the bispecific molecule of the composition described in any one of Embodiments 32 to 41.

[0290] Embodiment 43: A pharmaceutical composition comprising a bispecific molecule according to any one of Embodiments 1 to 31 or a composition according to any one of Embodiments 32 to 41, and a pharmaceutically acceptable carrier.

[0291] Embodiment 44: A method for treating an inflammatory disease or condition in a subject, comprising administering an effective amount of a bispecific molecule according to any one of Embodiments 1 to 31, a composition according to any one of Embodiments 32 to 41, or a pharmaceutical composition according to Embodiment 43 to the subject, thereby treating the disease or condition.

[0292] Embodiment 45: The method according to Embodiment 44, wherein the inflammatory disease or condition is associated with an increase in the activity and / or expression of TREM1, IL-17 family cytokines, IL-17R, one or more downstream inflammatory signaling proteins, or a combination thereof, compared to the activity and / or expression of TREM1, IL-17 family cytokines, IL-17R, one or more downstream inflammatory signaling proteins, or a combination thereof, in a subject prior to having the inflammatory disease or condition.

[0293] Embodiment 46: A method for treating psoriasis in a subject, comprising administering an effective amount of a bispecific molecule according to any one of Embodiments 1 to 31, a composition according to any one of Embodiments 32 to 41, or a pharmaceutical composition according to Embodiment 43 to the subject, thereby treating psoriasis.

[0294] Embodiment 47: The method according to Embodiment 46, which reduces the incidence of Candida infection in a subject compared to a subject treated with a monospecific antibody that reduces IL-17 activity.

[0295] Embodiment 48: A method for reducing an IL-17-related inflammatory state (or symptoms) in a subject, comprising administering an effective amount of a bispecific molecule according to any one of Embodiments 1 to 31, a composition according to any one of Embodiments 32 to 41, or a pharmaceutical composition according to Embodiment 43 to the subject, thereby reducing the IL-17-related inflammatory state (or symptoms) in the subject compared to the IL-17-related inflammatory state (or symptoms) in the subject before administration of the bispecific molecule.

[0296] Embodiment 49: A method for reducing a TREM1-related inflammatory state (or symptoms) in a subject, comprising administering an effective amount of a bispecific molecule according to any one of Embodiments 1 to 31, a composition according to any one of Embodiments 32 to 41, or a pharmaceutical composition according to Embodiment 43 to the subject, thereby reducing the TREM1-related inflammatory state (or symptoms) in the subject compared to the TREM1-related inflammatory state (or symptoms) in the subject before administration of the bispecific molecule.

[0297] Embodiment 50: Nicotinamide phosphoribosyltransferase (NAMPT), dehydrogenase / reductase 9 (DHRS9), cyclin-dependent kinase inhibitor 1A (CDKN1A), CD52 molecule (CD52), myotubularin-related protein 11 (MTMR11), EH domain-containing protein 1 (EHD1), solute transporter family 27 member 3 (SLC27A3), interleukin 24 (IL24), Pim-2 oncogene serine / threonine kinase (PIM2), chitinase 3-like protein 1 (CHI3L1), polypeptide N-acetylgalactosaminyltransferase 6 (GALNT6), acyl-CoA The method according to any one of embodiments 44 to 49, which increases the expression of at least one of the following: thioesterase 7 (ACOT7), cytokine-inducible SH2-containing protein (CISH), family containing sequence similarity 129 member A (FAM129A), polo-like kinase 3 (PLK3), major facilitator superfamily domain-containing 12 (MFSD12), StAR-related lipid transfer domain-containing 4 (STARD4), c-type lectin domain family 12 member A (CLEC12A), CD55 molecule (Cromer blood group) (CD55), and interferon-lambda receptor 1 (IFNLR1).

[0298] Embodiment 51: A method according to any one of Embodiments 44 to 50, for restoring the pentose phosphate pathway (PPP). [Examples]

[0299] (Example 1) Bispecific antibody construct A bispecific antibody capable of binding to two different targets is generated. The two different targets are (a) IL-17 and / or IL-17R, and (b) TREM1. The bispecific antibody comprises a first target-binding domain and a second target-binding domain. The first target-binding domain comprises a first VH sequence and a first VL sequence, where the first target-binding domain can bind to IL-17 and / or IL-17R. The second target-binding domain comprises a second VH sequence and a second VL sequence, where the second target-binding domain can bind to TREM1. The first VH sequence and the first VL sequence are selected according to one of the amino acid sequence combinations listed in Table 6. The second VH sequence comprises one of the amino acid sequences listed in Table 8. The second VL sequence comprises one of the amino acid sequences listed in Table 10. (Example 2) bispecific antibody Bispecific Matrix Generation

[0300] A total of eight different bispecificity candidates were prepared, each targeting IL17 and TREM1, respectively. Briefly, the nucleotide sequences encoding the first, second, and light chains were synthesized as shown in Table 24. Knob and hole techniques were used to design the constructs. The nucleotide sequences were codon-optimized. For control, two IL17-specific monoclonal antibodies, two TREM1-specific monoclonal antibodies, and six single-arm bispecificity controls were included in the design set. [Table 24]

[0301] A total of 18 heavy and light chain constructs were cloned using Gibson Assembly with the NEBuilder HiFi DNA Assembly Cloning Kit (NEB). After Gibson Assembly, the constructs were transformed using DH5a competent cells (NEB), seeded on LB-CARB trays (Teknova), and grown overnight at 37°C with shaking. Then, single colonies were picked from each construct on the LB-CARB tray for inoculation into LB-CARB medium (Teknova, freshly prepared) and seeded in 5 mL of LB-CARB medium in 15 mL Falcon tubes, grown overnight at 37°C with shaking. The cultured cell tubes were then pelleted, the medium discarded, and plasmid purification was initiated using the Qiagen plasmid plus midiprep purification kit (for yield) with a vacuum manifold and centrifugation. Plasmid DNA was then confirmed for concentration (on Nanodrop) and its nucleotide sequence was confirmed by Sanger sequencing. Dual-specific expression and purification

[0302] Proprietary variable heavy and light chain pairs were cloned into vectors designed to express bispecific antibodies and associated controls in HEK293 cells under the control of a CMV promoter. The antibody expression vectors were conjugated with polyethyleneimine and transfected into HEK293 cultures. During expression, enrichment for heterodimerization and exclusion for homodimerization were performed using knob and hole techniques. After shaking in 293 cell culture medium at 37°C for 5 days, the cultures were harvested, and the supernatant containing the secreted antibodies was clarified and filtered. Antibodies were captured from the supernatant via an agarose-based protein A resin on an FPLC. After several washes with PBS, the antibodies were eluted in phosphate and physiological saline solution (pH 3) and neutralized to approximately pH 5.5 with basic phosphate saline solution (pH 11). Analytical SEC and SDS-PAGE analysis were used to determine the optimal fraction derived from each protein A elution to proceed to secondary / polishing purification.

[0303] Samples were enriched with heterodimeric bispecific antibodies or controls using automated cation exchange chromatography (CEX) again on an FPLC, employing binding and elution methods. After dilution, the target material was captured in protein A elution using a strong cation exchange column (Capto S ImpACT) to reduce salt concentration, followed by washing and elution with sodium chloride at increasing levels at a constant pH of 6.5. A combination of analytical SEC and SDS-PAGE was used to identify fractions derived from CEX suitable for pooling. After pooling, samples were sterilized and assayed for purity by analytical SEC and for endotoxins by the Charles River LAL test. Bispecific activity

[0304] Human peripheral blood mononuclear cells (PBMCs) were used to determine the activity of the bispecific antibody. Briefly, PBMCs were stimulated for TREM1 activation using peptidoglycan-recognizing protein 1 (PGLYRP1) and peptidoglycan (PGN) at concentrations of 0.1 μg / ml and 0.3 μg / ml (1:3 ratio), respectively. Subsequently, the PBMCs were incubated with an anti-cytokine (anti-CD3) at a concentration of 0.5 μg / ml for T cell activation. The stimulated cells were then incubated with bispecific antibody candidate number 1 at a concentration of 50 nM. The cells were then centrifuged, and the supernatant was analyzed for the presence of IL-17 and tumor necrosis factor α (TNFα). The results for the presence of IL-17 and TNFα are shown in Figures 2A and 2B, respectively. Unstimulated cells were used as a negative control. Stimulated isotype cells were used as a control, and their activity was measured in the presence of an untargeted antibody containing an Fc silencing mutation similar to bispecific antibody candidate number 1. TREM1 antibody, IL-17 antibody, and combinations thereof were used as positive controls. (Example 3) Phase 1 proof-of-concept trial for bispecific antibodies

[0305] A bispecific antibody capable of binding to two different targets, IL-17 and / or IL-17R, and (b)TREM1, is generated as described in Example 1. The bispecific antibody is administered to a subject requiring it. The subject is selected according to the criteria listed in Table 25. [Table 25]

Claims

1. A bispecific molecule comprising a first domain and a second domain, wherein the first domain binds to TREM1 or a functional fragment thereof, and the second domain binds to IL-17, IL-17R, or a functional fragment thereof.

2. The bispecific molecule according to claim 1, which is an antibody, an antibody variant, or an engineered functional fragment of an antibody.

3. The bispecific molecule according to claim 1 or 2, wherein the IL-17 comprises one of IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, IL-17F, IL-17A / F, a combination thereof, or a functional fragment.

4. A bispecific molecule according to any one of claims 1 to 3, wherein the binding affinity of the first domain to TREM1 is lower than the binding affinity of the second domain to IL-17, IL-17R, or a functional fragment thereof.

5. The bispecific molecule according to claim 4, wherein the binding affinity of the second binding domain to IL-17, IL-17R, or a functional fragment thereof is at least twice the binding affinity of the first domain to TREM1.

6. The bispecific molecule according to any one of claims 1 to 3, wherein the binding affinity of the first domain to TREM1 is higher than the binding affinity of the second domain to IL-17, IL-17R, or a functional fragment thereof.

7. The bispecific molecule according to claim 6, wherein the binding affinity of the first domain to TREM1 is at least twice the binding affinity of the second binding domain to IL-17, IL-17R, or a functional fragment thereof.

8. A bispecific molecule according to any one of claims 1 to 7, which is a Fab2 antibody, a bis-scFv antibody, a diabody, DVD-Ig, TandAb, tandem scFv-Fc, one-arm tandem scFv-Fc, DART, DART-Fc, or a functional fragment thereof.

9. The bispecific molecule according to any one of claims 1 to 8, wherein the first domain comprises a TREM1-binding heavy chain variable domain.

10. The bispecific molecule according to any one of claims 1 to 9, wherein the first domain comprises a TREM1-binding light chain variable domain.

11. The bispecific molecule according to any one of claims 1 to 10, wherein the second domain comprises an IL-17 binding heavy chain variable domain.

12. The bispecific molecule according to any one of claims 1 to 11, wherein the second domain comprises an IL-17 binding light chain variable domain.

13. The bispecific molecule according to any one of claims 1 to 12, wherein at least one of the first domain and the second domain comprises a light chain constant domain and / or a heavy chain constant domain.

14. A bispecific molecule according to any one of claims 1 to 13, comprising at least one of an Fc region and / or a Fab region.

15. The bispecific molecule according to claim 14, wherein the Fc region includes an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to any one of the amino acid sequences of SEQ ID NOs: 199, 722, and 723.

16. The bispecific molecule according to claim 14, wherein the heavy chain constant domain of the first domain includes an Fc region having S354C mutation and T366W mutation in EU numbering, and the heavy chain constant domain of the second domain includes an Fc region having Y349C mutation, T366S mutation and Y407V mutation in EU numbering.

17. The bispecific molecule according to claim 14, wherein the heavy chain constant domain of the second domain includes an Fc region having S354C mutation and T366W mutation in EU numbering, and the heavy chain constant domain of the first domain includes an Fc region having Y349C mutation, T366S mutation and Y407V mutation in EU numbering.

18. The bispecific molecule according to claim 14, wherein the Fc region comprises a human IgG1 heavy chain constant chain having at least one substitution selected from the EU numbering positions N297, C226, C229, E233, L234, L235, G236, G237, P238, F243, M252, S254, T256, D265, S267, H268, D270, P271, R292, Y300, K322, A327, L328, P329, A330, P331, and P396.

19. The bispecific molecule according to claim 14, wherein the Fc region comprises a human IgG2 heavy chain constant chain having at least one substitution selected from the EU numbering positions C232, C233, V234, G237, P238, M252, S254, T256, H268, N297, V309, A330, and P331.

20. The bispecific molecule according to claim 14, wherein the Fc region comprises a human IgG4 heavy chain constant chain having at least one substitution selected from EU numbering positions S228, E233, F234, L235, L236, G237, S241, L248, M252, S254, T256, N297, E318, and T394.

21. A bispecific molecule according to any one of claims 1 to 20, comprising at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, or higher anti-inflammatory activity compared to a combination of a monospecific antibody that binds to TREM1 and a monospecific antibody that binds to a cytokine of the IL-17 family, IL-17R, or a combination thereof.

22. A composition comprising the bispecific molecule according to any one of claims 1 to 21.

23. A composition comprising a bispecific molecule having a first domain and a second domain, The first domain binds to TREM1 or a functional fragment thereof. The second domain is bound to IL-17, IL-17R or a functional fragment thereof. A composition wherein administration of an effective amount of the composition to a subject requiring it results in the treatment of an inflammatory disease or condition.

24. The composition according to claim 23, wherein IL-17 includes IL-17A, IL-17A / F, or any combination thereof.

25. The composition according to claim 23 or 24, wherein the inflammatory disease or condition is selected from the group consisting of rheumatoid arthritis, juvenile arthritis, psoriatic arthritis, ankylosing spondylitis, psoriasis, sweat gland abscess, ulcerative colitis, Crohn's disease, necrotizing enterocolitis, sepsis, axial spondyloarthritis, or multiple sclerosis.

26. The composition according to claim 23 or 24, wherein the inflammatory disease or condition is rheumatoid arthritis, juvenile arthritis, psoriatic arthritis, axial spondyloarthritis, or ankylosing spondylitis.

27. The composition according to claim 23 or 24, wherein the inflammatory disease or condition is psoriasis or sweat gland abscess.

28. The composition according to claim 23 or 24, wherein the inflammatory disease or condition is ulcerative colitis, Crohn's disease, necrotizing enterocolitis, sepsis, or multiple sclerosis.

29. The composition according to claim 23 or 24, wherein the inflammatory disease or condition is sepsis.

30. The composition according to claim 23 or 24, wherein the inflammatory disease or condition is multiple sclerosis.

31. A nucleic acid encoding at least a portion of the bispecific molecule according to any one of claims 1 to 21 or the bispecific molecule of the composition according to any one of claims 22 to 30.

32. A pharmaceutical composition comprising a bispecific molecule according to any one of claims 1 to 21 or a composition according to any one of claims 22 to 30, and a pharmaceutically acceptable carrier.

33. A method for treating an inflammatory disease or condition in a subject, comprising administering to the subject an effective amount of a bispecific molecule according to any one of claims 1 to 21, a composition according to any one of claims 22 to 30, or a pharmaceutical composition according to claim 32, thereby treating the inflammatory disease or condition.

34. The method according to claim 33, wherein the inflammatory disease or condition is associated with increased activity and / or expression of TREM1, IL-17 family cytokines, IL-17R, one or more downstream inflammatory signaling proteins, or combinations thereof, compared to a subject without the inflammatory disease or condition.

35. A method for treating psoriasis in a subject, comprising administering to the subject an effective amount of a bispecific molecule according to any one of claims 1 to 21, a composition according to any one of claims 22 to 30, or a pharmaceutical composition according to claim 32, thereby treating psoriasis.

36. The method according to claim 35, which reduces the incidence of Candida infection in the subject compared to the subject treated with a monospecific antibody that reduces IL-17 activity.

37. A method for reducing an IL-17-related inflammatory state in a subject, comprising administering to the subject an effective amount of a bispecific molecule according to any one of claims 1 to 21, a composition according to any one of claims 22 to 30, or a pharmaceutical composition according to claim 32, thereby reducing the IL-17-related inflammatory state in the subject compared to the IL-17-related inflammatory state in the subject before administration of the bispecific molecule.

38. A method for reducing the TREM1-related inflammatory state in a subject, comprising administering an effective amount of a bispecific molecule according to any one of claims 1 to 21, a composition according to any one of claims 22 to 30, or a pharmaceutical composition according to claim 32 to the subject, thereby reducing the TREM1-related inflammatory state in the subject compared to the TREM1-related inflammatory state in the subject before administration of the bispecific molecule.

39. Nicotinamide phosphoribosyltransferase (NAMPT), dehydrogenase / reductase 9 (DHRS9), cyclin-dependent kinase inhibitor 1A (CDKN1A), CD52 molecule (CD52), myotubularin-related protein 11 (MTMR11), EH domain-containing protein 1 (EHD1), solute transporter family 27 member 3 (SLC27A3), interleukin 24 (IL24), Pim-2 oncogene serine / threonine kinase (PIM2), chitinase 3-like protein 1 (CHI3L1), polypeptide N-acetylgalactosaminyltransferase 6 (GALNT6), acyl-CoA thioe The method according to any one of claims 33 to 38, which increases the expression of at least one of the following: sterase 7 (ACOT7), cytokine-inducible SH2-containing protein (CISH), sequence-similarity 129 member A-containing family (FAM129A), polo-like kinase 3 (PLK3), major facilitator superfamily domain-containing 12 (MFSD12), StAR-related lipid transfer domain-containing 4 (STARD4), c-type lectin domain family 12 member A (CLEC12A), CD55 molecule (Cromer blood group) (CD55), and interferon-lambda receptor 1 (IFNLR1).

40. The method according to any one of claims 33 to 39, for restoring the pentose phosphate pathway (PPP).

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