Engineered bispecific molecules and methods of use

Abstract

Provided herein are engineered proteins and methods of treatment using the engineered proteins wherein the engineered proteins comprise a first region and a second region wherein the first region binds to TREM1, a variant thereof, or a functional fragment thereof, and wherein the second region binds to interleukin, such as TREM1, a variant thereof, or a functional fragment thereof. Wherein the interleukin comprises a protein selected from any one of the IL-1 family, the IL-6 family, the IL-12 family and the IL-23 family, a variant thereof, or a functional fragment thereof.

Description

Cross-reference application

[0001] This application claims priority to U.S. Provisional Application No. 63 / 518,463, filed August 9, 2023, the entire contents of each of which are incorporated herein by reference. By referencing and incorporating into the sequence list

[0002] This application includes a sequence list submitted via the Patent Centre. The sequence list with the title 220710-702601_PCT_SL.xml, created on August 8, 2024 and measuring 908,921 bytes, is hereby incorporated in its entirety by reference. Technical Field

[0003] This disclosure generally relates to engineered protein molecules that bind TREM1 and interleukins. Background Technology

[0004] Bispecific antibodies (BsAbs) are antibodies that independently target two different antigens or optionally two different epitopes on the same antigen. Compared to monospecific antibodies, BsAbs have shown the potential for enhanced therapeutic activity. It is understood that BsAbs have a wider range of applications in immunotherapy for treating various diseases. Invention Overview

[0005] Engineered protein constructs that combine both TREM1 and interleukins are provided, along with other exemplary compositions. For example, in some embodiments, the engineered protein construct (such as a bispecific molecule) comprises a first region and a second region. In some embodiments, the first region binds TREM1, a variant thereof, or a functional fragment thereof. In some embodiments, the second region binds interleukins, wherein the interleukin comprises a protein, a variant thereof, or a functional fragment thereof selected from the IL-1 family, the IL-6 family, the IL-12 family, and the IL-23 family. In some embodiments, the engineered protein construct is an antibody, a variant thereof, or a functional fragment thereof. In some embodiments, the engineered protein construct is a Fab2 antibody, a dual scFv antibody, a dual antibody, DVD-Ig, TandAb, a tandem scFv-Fc, a single-arm tandem scFv-Fc, DART, DART-Fc, or a functional fragment thereof. In some embodiments, the engineered protein construct comprises a heterodimeric antibody or a functional fragment thereof. In some embodiments, the engineered protein construct comprises a constant region. In some embodiments, the first region comprises a TREM1-binding heavy chain variable domain. In some embodiments, the first region comprises a TREM1-binding light chain variable domain. In some embodiments, the second region comprises an interleukin-binding heavy chain variable domain. In some embodiments, the second region comprises an interleukin-binding light chain variable domain. In some embodiments, the binding affinity of the first region to TREM1 is lower than the binding affinity of the second region to interleukin. In some embodiments, the binding affinity of the second region to interleukin is at least twice that of the first region to TREM1. In some embodiments, the binding affinity of the first region to TREM1 is higher than that of the second region to interleukin. In some embodiments, the binding affinity of the first region to TREM1 is at least twice that of the second region to interleukin. In some embodiments, at least one of the first and second regions comprises a light chain constant domain and / or a heavy chain constant domain. In some embodiments, the engineered protein construct comprises at least one of an Fc region and / or a Fab region. In some embodiments, the Fc region comprises 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 amino acid sequences to any one of the amino acid sequences of SEQ ID NO: 453-455. In some embodiments, the heavy chain constant domain of the first region comprises an Fc region having the S354C mutation and the T366W mutation according to EU numbers, and the heavy chain constant domain of the second region comprises an Fc region having the Y349C mutation, the T366S mutation, and the Y407V mutation according to EU numbers.In some embodiments, the heavy chain constant domain of the second region includes an Fc region having an S354C mutation and a T366W mutation according to EU numbers, and the heavy chain constant domain of the first region includes an Fc region having an Y349C mutation, a T366S mutation, and a Y407V mutation according to EU numbers. In some embodiments, the Fc region includes a human IgG1 heavy chain constant with at least one substitution selected from 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 according to EU numbers. In some embodiments, the Fc region comprises a human IgG2 heavy chain constant with at least one substitution selected from positions C232, C233, V234, G237, P238, M252, S254, T256, H268, N297, V309, A330, and P331 according to EU numbers. In some embodiments, the Fc region comprises a human IgG4 heavy chain constant with at least one substitution selected from positions S228, E233, F234, L235, L236, G237, S241, L248, M252, S254, T256, N297, E318, and T394 according to EU numbers. In some embodiments, the engineered protein constructs described herein exhibit pH-dependent target-binding activity to a target peptide selected from TREM1, interleukins, their variants, and functional fragments thereof, and wherein interleukins are selected from the IL-1 family, IL-6 family, IL-12 family, and IL-23 family proteins. In some embodiments, the engineered protein constructs comprise a combined anti-inflammatory activity of 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, relative to the combined anti-inflammatory activity of a monospecific antibody binding TREM1 and a monospecific antibody binding interleukins.

[0006] This article also describes compositions that include the engineered protein constructs described herein.

[0007] This document also describes engineered protein constructs for use in the treatment of inflammatory diseases or conditions, wherein the engineered protein construct is any of the engineered protein constructs described herein. In some embodiments, the inflammatory disease or condition is selected from the group consisting of: rheumatoid arthritis, juvenile arthritis, psoriatic arthritis, ankylosing spondylitis, axial spondyloarthritis, psoriasis, hidradenitis suppurativa, ulcerative colitis, Crohn's disease, necrotizing enterocolitis, sepsis, or multiple sclerosis.

[0008] This article also describes pharmaceutical compositions comprising any of the engineered protein constructs described herein (e.g., bispecific) and a pharmaceutically acceptable carrier.

[0009] This document also describes pharmaceutical compositions for use in the treatment of inflammatory diseases or conditions, wherein the pharmaceutical composition comprises: a TREM1 binding moiety, an interleukin binding moiety, and a pharmaceutically acceptable carrier, wherein the interleukin binding moiety comprises a protein selected from IL-1 binding moieties, IL-6 binding moieties, IL-12 binding moieties, and IL-23 binding moieties, and wherein administration of an effective amount of the composition to a subject with appropriate need results in treatment of the inflammatory disease or condition. In some embodiments, the inflammatory disease or condition is selected from the group consisting of: rheumatoid arthritis, juvenile arthritis, psoriatic arthritis, ankylosing spondylitis, axial spondyloarthritis, psoriasis, hidradenitis suppurativa, ulcerative colitis, Crohn's disease, necrotizing enterocolitis, sepsis, or multiple sclerosis.

[0010] This document also describes methods for treating inflammatory diseases or conditions in subjects. In some embodiments, the methods include administering to the subject an effective amount of the engineered protein construct, composition, or pharmaceutical composition described herein, 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, interleukins, one or more of their downstream inflammatory signaling proteins, or combinations thereof, relative to subjects without the inflammatory disease or condition. In some embodiments, the methods reduce the incidence of Candida infection in subjects relative to subjects treated with monospecific antibodies that reduce interleukin activity.

[0011] This document also describes compositions. In some embodiments, the composition comprises an engineered protein construct comprising a first region and a second region, wherein the first region binds TREM1, a variant thereof, or a functional fragment thereof, wherein the second region binds interleukins, wherein the interleukins comprise proteins, variants thereof, or functional fragments thereof selected from the IL-1 family, the IL-6 family, the IL-12 family, and the IL-23 family, and wherein administration of an effective amount of the composition to a subject with appropriate need results in treatment of an inflammatory disease or condition. In some embodiments, the inflammatory disease or condition is selected from the group consisting of: rheumatoid arthritis, juvenile arthritis, psoriatic arthritis, ankylosing spondylitis, axial spondyloarthritis, psoriasis, hidradenitis suppurativa, 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 spondyloarthritis, or ankylosing spondylitis. In some embodiments, the inflammatory disease or condition is psoriasis or hidradenitis suppurativa. 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.

[0012] This document also describes nucleic acids. In some embodiments, the nucleic acid encodes any of the engineered protein constructs described herein (e.g., multispecific molecules (e.g., bispecific molecules)) or at least a portion of an engineered protein construct of a composition described herein.

[0013] This article also describes a method for reducing IL-1-related inflammatory status in subjects, comprising administering to the subject an effective amount of a pharmaceutical composition comprising an IL-1-binding moiety, a TREM1-binding moiety, and a pharmaceutically acceptable carrier, thereby reducing the subject's IL-1-related inflammatory status relative to the subject's IL-1-related inflammatory status prior to administration of the pharmaceutical composition. In some embodiments, the method increases the expression of at least one of the following: nicotinamide phosphoribosyltransferase (NAMPT), dehydrogenase / reductase 9 (DHRS9), cyclin-dependent kinase inhibitor 1A (CDKN1A), CD52 molecule (CD52), myotubulin-associated protein 11 (MTMR11), EH domain-containing 1 (EHD1), solute carrier family 27 member 3 (SLC27A3), interleukin 24 (IL24), Pim-2 proto-oncogene serine / threonine kinase (PIM2), chitosanase 3-like 1 (CHI3L1), peptide N-acetylgalactosyltransferase 6 (GALNT6), acyl-CoA thioesterase 7 (ACOT7), protein containing cytokine-inducible SH2 (CISH), sequence-similar family 129 member A (FAM129A), polo-like kinase 3 (PLK3), major facilitater superfamily domain 12 (MFSD12), and StAR-associated lipid transfer domain 4. (STARD4), C-type lectin domain family 12 member A (CLEC12A), CD55 molecule (Cromer blood group) (CD55), and interferon λ receptor 1 (IFNLR1). In some embodiments, the method restores the pentose phosphate pathway (PPP). In some embodiments, the pharmaceutical composition comprises any of the pharmaceutical compositions described herein.

[0014] This article also describes a method for reducing IL-6-related inflammatory status in subjects, comprising administering to the subject an effective amount of a pharmaceutical composition comprising an IL-6-binding moiety, a TREM1-binding moiety, and a pharmaceutically acceptable carrier, thereby reducing the subject's IL-6-related inflammatory status relative to the subject's IL-6-related inflammatory status before administration of the pharmaceutical composition. In some embodiments, the method increases the expression of at least one of the following: nicotinamide phosphoribosyltransferase (NAMPT), dehydrogenase / reductase 9 (DHRS9), cyclin-dependent kinase inhibitor 1A (CDKN1A), CD52 molecule (CD52), myotubulin-associated protein 11 (MTMR11), EH domain-containing 1 (EHD1), solute carrier family 27 member 3 (SLC27A3), interleukin 24 (IL24), Pim-2 proto-oncogene serine / threonine kinase (PIM2), chitosanase 3-like 1 (CHI3L1), peptide N-acetylgalactosyltransferase 6 (GALNT6), acyl-CoA thioesterase 7 (ACOT7), protein containing cytokine-inducible SH2 (CISH), sequence-similar family 129 member A (FAM129A), polo-like kinase 3 (PLK3), major facilitater superfamily domain 12 (MFSD12), and StAR-associated lipid transfer domain 4. (STARD4), C-type lectin domain family 12 member A (CLEC12A), CD55 molecule (Cromer blood group) (CD55), and interferon λ receptor 1 (IFNLR1). In some embodiments, the method restores the pentose phosphate pathway (PPP). In some embodiments, the pharmaceutical composition comprises any of the pharmaceutical compositions described herein.

[0015] This article also describes a method for reducing IL-12-related inflammatory status in subjects, comprising administering to the subject an effective amount of a pharmaceutical composition comprising an IL-12-binding moiety, a TREM1-binding moiety, and a pharmaceutically acceptable carrier, thereby reducing the subject's IL-12-related inflammatory status relative to the subject's IL-12-related inflammatory status prior to administration of the pharmaceutical composition. In some embodiments, the method increases the expression of at least one of the following: nicotinamide phosphoribosyltransferase (NAMPT), dehydrogenase / reductase 9 (DHRS9), cyclin-dependent kinase inhibitor 1A (CDKN1A), CD52 molecule (CD52), myotubule-associated protein 11 (MTMR11), EH domain-containing 1 (EHD1), solute carrier family 27 member 3 (SLC27A3), interleukin 24 (IL24), Pim-2 proto-oncogene serine / threonine kinase (PIM2), chitosanase 3-like 1 (CHI3L1), polypeptide N-acetylgalactosyltransferase 6 (GALNT6), acyl-CoA thioesterase 7 (ACOT7), protein containing cytokine-inducible SH2 (CISH), sequence-similar family 129 member A (FAM129A), polo-like kinase 3 (PLK3), and protein containing major facilitater superfamily 12. (MFSD12), containing StAR-associated lipid transfer domain 4 (STARD4), c-type lectin domain family 12 member A (CLEC12A), CD55 molecule (Cromer blood group) (CD55), and interferon λ receptor 1 (IFNLR1). In some embodiments, the method restores the pentose phosphate pathway (PPP). In some embodiments, the pharmaceutical composition comprises any of the pharmaceutical compositions described herein.

[0016] This article also describes a method for reducing IL-23-related inflammatory status in subjects, comprising administering to the subject an effective amount of a pharmaceutical composition comprising an IL-23-binding moiety, a TREM1-binding moiety, and a pharmaceutically acceptable carrier, thereby reducing the subject's IL-23-related inflammatory status relative to the subject's IL-23-related inflammatory status prior to administration of the pharmaceutical composition. In some embodiments, the method increases the expression of at least one of the following: nicotinamide phosphoribosyltransferase (NAMPT), dehydrogenase / reductase 9 (DHRS9), cyclin-dependent kinase inhibitor 1A (CDKN1A), CD52 molecule (CD52), myotubule-associated protein 11 (MTMR11), EH domain-containing 1 (EHD1), solute carrier family 27 member 3 (SLC27A3), interleukin 24 (IL24), Pim-2 proto-oncogene serine / threonine kinase (PIM2), chitosanase 3-like 1 (CHI3L1), polypeptide N-acetylgalactosyltransferase 6 (GALNT6), acyl-CoA thioesterase 7 (ACOT7), protein containing cytokine-inducible SH2 (CISH), sequence-similar family 129 member A (FAM129A), polo-like kinase 3 (PLK3), and protein containing major facilitater superfamily 12. (MFSD12), containing StAR-associated lipid transfer domain 4 (STARD4), c-type lectin domain family 12 member A (CLEC12A), CD55 molecule (Cromer blood group) (CD55), and interferon λ receptor 1 (IFNLR1). In some embodiments, the method restores the pentose phosphate pathway (PPP). In some embodiments, the pharmaceutical composition comprises any of the pharmaceutical compositions described herein.

[0017] This article also describes a method for reducing TREM1-related inflammatory status in subjects, comprising administering to subjects an effective amount of a pharmaceutical composition comprising a TREM1-binding moiety, an interleukin-binding moiety, and a pharmaceutically acceptable carrier, thereby reducing the subjects' TREM1-related inflammatory status relative to the TREM1-related inflammatory status prior to administration of the pharmaceutical composition. In some embodiments, the method increases the expression of at least one of the following: nicotinamide phosphoribosyltransferase (NAMPT), dehydrogenase / reductase 9 (DHRS9), cyclin-dependent kinase inhibitor 1A (CDKN1A), CD52 molecule (CD52), myotubule-associated protein 11 (MTMR11), EH domain-containing 1 (EHD1), solute carrier family 27 member 3 (SLC27A3), interleukin 24 (IL24), Pim-2 proto-oncogene serine / threonine kinase (PIM2), chitosanase 3-like 1 (CHI3L1), polypeptide N-acetylgalactosyltransferase 6 (GALNT6), acyl-CoA thioesterase 7 (ACOT7), protein containing cytokine-inducible SH2 (CISH), sequence-similar family 129 member A (FAM129A), polo-like kinase 3 (PLK3), and protein containing major facilitater superfamily 12. (MFSD12), containing StAR-associated lipid transfer domain 4 (STARD4), c-type lectin domain family 12 member A (CLEC12A), CD55 molecule (Cromer blood group) (CD55), and interferon λ receptor 1 (IFNLR1). In some embodiments, the method restores the pentose phosphate pathway (PPP). In some embodiments, the pharmaceutical composition comprises any of the pharmaceutical compositions described herein. By incorporating via reference

[0018] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the extent that each individual publication, patent or patent application is specifically and individually indicated to be incorporated by reference. Brief description of the attached diagram

[0019] The features of this disclosure are specifically set forth in the appended claims. A better understanding of the features and advantages of this disclosure will be obtained by referring to the following detailed description of illustrative embodiments utilizing the principles of this disclosure, along with the accompanying drawings, in which: Figure 1 A bispecific antibody containing both a TREM1 binding region and an interleukin binding region was described.

[0020] Figure 2The effect of contacting human peripheral blood mononuclear cells (PBMCs) with an antibody combination is demonstrated, wherein the combination of TREM1-binding antibody and interleukin (e.g., IL-6 and IL-23)-binding antibody is shown. In short, Figure 2 The amounts of tumor necrosis factor α (TNFα), IL-1β, IL-17, IL-23, and macrophage inflammatory protein-3α (MIP-3α) present in the supernatant of human PBMCs after combined exposure to TREM1-binding antibody and interleukin-binding antibody (IL-6 or IL-23) are shown. Detailed Implementation

[0021] The following description and examples illustrate embodiments of this disclosure in detail. It should be understood that this disclosure is not limited to the specific embodiments described herein and therefore can be varied. Those skilled in the art will recognize that many variations and modifications are possible with respect to this disclosure, all of which are included within the scope of this invention.

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

[0023] The chapter titles used in this article are for organizational purposes only and are not to be construed as limiting the topics described.

[0024] Although the various features of this disclosure may be described in the context of a single embodiment, such features may also be provided individually or in any suitable combination. Conversely, although this disclosure may be described in the context of a single embodiment herein for clarity, this disclosure may also be implemented in a single embodiment.

[0025] definition The following definitions supplement those in the art and are specific to this application, and should not be attributed to any related or unrelated circumstances, such as any jointly owned patents or applications. While any methods and materials similar to or equivalent to those described herein may be used in the practice of testing the contents of this disclosure, preferred materials and methods are described herein. Therefore, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be restrictive.

[0026] In this application, unless otherwise expressly stated, the use of the singular includes the plural. It must be noted that, unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” as used in the specification include the plural reference. In this application, unless otherwise stated, the use of “or” means “and / or.” Furthermore, the use of the term “including” and other forms such as “include,” “includes,” and “included” is not restrictive.

[0027] The references in the specification to “some implementation schemes,” “implementation schemes,” “an implementation scheme,” or “other implementation schemes” mean that a particular feature, structure, or characteristic described in connection with an implementation scheme is included in at least some of the implementation schemes of this disclosure, but not necessarily in all of them.

[0028] As used in this specification and claims, the terms "comprising" (and any form of "comprising," such as "comprise" and "comprises"), "having" (and any form of "having," such as "have" and "has"), "including" (and any form of "including," such as "includes" and "include"), or "containing" (and any form of "containing," such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unlisted elements or method steps. It is contemplated that any embodiments discussed in this specification can be implemented with respect to any method or composition of this disclosure, and vice versa. Furthermore, the compositions of this disclosure can be used to implement the methods of this disclosure.

[0029] The term "about" or "approximately" means within an acceptable range of error for a particular value, as determined by a person skilled in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" may, according to practice in the art, mean within one or more standard deviations. Alternatively, "about" may mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. In another instance, the quantity "about 10" includes 10 and any quantity from 9 to 11. In yet another instance, the term "about" with respect to a reference value may also include a range of values ​​plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%. Alternatively, particularly for biological systems or processes, the term "about" may mean within an order of magnitude of the value, preferably within five times the value, and more preferably within two times the value. When a particular value is described in this application and claims, unless otherwise stated, the term “about” shall be assumed to mean an acceptable range of error for the particular value.

[0030] As used herein, the terms “disease,” “disorder,” and “condition,” which may be used interchangeably herein, refer to any alteration of state in which the body or organs are disrupted or interfere with the functioning of the body, and / or cause symptoms in the person afflicted or in contact with the person afflicted, such as discomfort, dysfunction, pain, or even death. Disease or disorder may also be associated with fever, ailing, ailment, malady, disorder, sickness, illness, complaint, or affectation.

[0031] As used herein, when used in the context of therapeutic or preventative treatment, the term "having a corresponding need" means having a disease, being diagnosed with a disease, or needing to prevent a disease, for example, for someone at risk of developing a disease. Therefore, a subject with a corresponding need can be a subject who needs treatment or prevention for a disease.

[0032] As used herein, the term "administration" means placing a compound (e.g., an antibody or antigen-binding fragment thereof, as disclosed herein) into a subject by a method or route that results in at least partial delivery of the agent at a desired site. The pharmaceutical compositions disclosed herein comprising antibodies or antigen-binding fragments thereof may be administered via any suitable route that results in effective treatment in a subject, including but not limited to intravenous injection, intra-arterial injection, subcutaneous injection, or direct infusion into tissue parenchyma. Where necessary or desired, administration may include, for example, intraventricular (“ICV”) administration, intranasal administration, intracranial administration, intracerebral administration, intracerebellar administration, subcutaneous administration, or intrathecal administration.

[0033] As used herein, the interchangeable terms “subject,” “patient,” “individual,” and similar terms refer to a vertebrate, mammal, primate, or human. Mammals include, but are not limited to, humans, primates, rodents, wild animals, or domesticated animals, including feral animals, farm animals, sporting animals, and pets. Primates include, for example, chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, such as rhesus monkeys. Rodents include, for example, mice, rats, marmots, ferrets, rabbits, and hamsters. Domesticated and game animals include, for example, cattle, horses, pigs, deer, bison, buffalo, feline species (e.g., domestic cats) and canine species (e.g., dogs, foxes, wolves), bird species (e.g., chickens, emus, ostriches), and fish (e.g., trout, catfish, and salmon). The terms “individual,” “patient,” and “subject” are used interchangeably herein. A subject can be male or female. In some embodiments, the subject is a mammal. Mammals can be humans, non-human primates, mice, rats, dogs, cats, horses, or cattle, but are not limited to these examples. Mammals other than humans can be advantageously used as subjects in animal models representing the condition or disorder. Non-limiting examples include murine models. Furthermore, the compositions and methods described herein can be used to treat domestic animals and / or pets. Subjects can be subjects diagnosed with a specific disorder and currently receiving treatment, or seeking treatment, monitoring, adjustment, or modification of existing therapeutic treatment, or subjects at risk of developing a specific disorder.

[0034] As used herein, the interchangeable terms “protein,” “peptide,” and “polypeptide” refer to a series of amino acid residues linked together by peptide bonds between adjacent α-amino and carboxyl groups. The terms “protein,” “peptide,” and “polypeptide” refer to polymers of amino acids (including modified amino acids (e.g., phosphorylated, glycosylated, glycosylated, etc.) and amino acid analogs), regardless of their size or function. “Protein” and “polypeptide” are generally used to refer to relatively large polypeptides, while the term “peptide” is generally used to refer to small polypeptides; however, the use of these terms overlaps in the art. The terms “protein,” “peptide,” and “polypeptide” are used interchangeably herein when referring to gene products and fragments thereof. These terms include, for example, natural and artificial proteins, polypeptide analogs of protein fragments and protein sequences (such as mutant proteins, variants, and fusion proteins), and proteins that are post-translational modified or otherwise covalently or non-covalently modified. Peptides, polypeptides, or proteins can be monomers or polymers. Polypeptides can have the amino acid sequence of a naturally occurring polypeptide from any mammal. Such naturally occurring sequence 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 bioactive polypeptide that, after aligning the sequence and introducing vacancies (if necessary) to achieve the maximum percentage of sequence identity, and without considering any conserved substitutions as part of the sequence identity, has at least about 80% amino acid sequence identity with the native sequence polypeptide. Such variants include, for example, polypeptides in which one or more amino acid residues are added or deleted at the N-terminus or C-terminus of the polypeptide. In some embodiments, the variant will have at least about 80% amino acid sequence identity. In some embodiments, the variant will have at least about 90% amino acid sequence identity. In some embodiments, the variant will have at least about 95% amino acid sequence identity with the native sequence polypeptide. A "derivative" of the polypeptide is a polypeptide that has been chemically modified (e.g., an antibody), said chemical modification being, for example, via conjugation to another chemical moiety (such as, for example, polyethylene glycol or albumin, e.g., human serum albumin), phosphorylation, and glycosylation.

[0035] As used herein, in the context of two or more nucleic acid or polypeptide sequences, the term "percentage of identity" refers to the percentage of identical nucleotide or amino acid residues in two or more sequences or subsequences when compared and aligned for maximum correspondence, as measured by 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 by visual inspection. Software used 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 across the full length of the sequences being compared. Depending on the application, the "percentage of identity" may exist within a region of the compared sequences, for example, within a functional domain, or, alternatively, across the full length of the two sequences being compared. For sequence comparisons, typically one sequence is used as a reference sequence against which the test sequence is compared. When using sequence comparison algorithms, the test and reference sequences are input into the computer, and subsequence coordinates are specified if necessary, along with the sequence algorithm program parameters. The sequence comparison algorithm then calculates the percentage of sequence identity between the test sequence and the reference sequence based on the specified program parameters. The optimal alignment of the sequences for comparison can be performed, for example, by the local 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 retrieval method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by the computerized implementation of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin genetic software package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (generally see Ausubel et al., above).

[0036] As used herein, the terms “increased,” “increase,” and “enhance” refer to an increase that is statistically significant; for the avoidance of doubt, the terms “increased,” “increase,” or “enhance” mean an increase of at least 10% compared to a reference level, such as 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 up to and including 100%, or any increase between 10% and 100%, or 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 and 10 times or greater compared to a reference level.

[0037] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to or is immunoreactive to a particular antigen, which in the present examples may be, for example, TREM1, the IL-1 family, the IL-6 family, the IL-12 family, or the IL-23 family. Antibodies may include, for example, polyclonal antibodies, monoclonal antibodies, genetically engineered antibodies, and their antigen-binding fragments. Antibodies may be, for example, mouse antibodies, chimeric antibodies, humanized antibodies, heteroconjugated antibodies, bispecific antibodies, 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 antibodies, V... HH V NARMonoclonal antibodies, sdAbs, or nanobodies. As used herein, the term "monoclonal antibody" refers to an antibody produced by a single clone of a B cell and binding to the same epitope. In contrast, "polyclonal antibody" refers to a group of antibodies produced by different B cells and binding to different epitopes of the same antigen. A complete antibody may comprise 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 an N-terminal variable (VH) region and three C-terminal constant (CH1, CH2, and CH3) regions, and each light chain may contain an N-terminal variable (VL) region and a 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, more than one distinct antigen-binding site may be present. The VH and VL regions may have similar general structures, wherein each region contains four frame regions whose sequences are relatively conserved. In some embodiments, the frame regions may be linked by three complementarity-determining regions (CDRs). In some implementations, the three CDRs, referred to as CDR1, CDR2, and CDR3, form the "hypervariate region" of the antibody, which is 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 is derived from a particular source or species, while the remainder of the heavy chain and / or light chain is derived from a different source or species.

[0039] As used herein, the term "human antibody" refers to an antibody that contains an amino acid sequence corresponding to the amino acid sequence of an antibody produced by humans or human cells, or is derived from a non-human source (e.g., obtained from a human source or designed de novo) using a human antibody library or human antibody encoding sequence.

[0040] As used herein, the term "humanized antibody" refers to an amino acid sequence in which one or more amino acids are substituted, deleted, and / or added that differ from the amino acid sequence of an antibody derived from a non-human species, such that when administered to a human subject, the humanized antibody is less likely to induce an immune response and / or induce a less severe immune response compared to a non-human species antibody. In some embodiments, certain amino acids in the framework and constant domains of the heavy and / or light chains of a non-human species antibody are mutated to produce a humanized antibody. In some embodiments, one or more constant domains from a human antibody are fused to one or more variable domains from a non-human species. In another embodiment, one or more amino acid residues in one or more CDR sequences of a non-human antibody are altered to reduce its potential immunogenicity when administered to a human subject, wherein the altered amino acid residues are not important for the immune-specific binding of the antibody to its antigen, or the alteration to the amino acid sequence is a conserved alteration such that 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 how to generate 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, each of the references is incorporated in its entirety by reference.

[0041] As used herein, the term "epitope" refers to the portion of an antigen that specifically binds to an antibody. Epitopes typically consist of surface-accessible amino acid residues and / or sugar side chains and may possess specific three-dimensional structural features and specific charge characteristics. The difference between conformational and non-conformational epitopes is that binding to the former, but not the latter, can be lost in the presence of denaturing solvents. Epitopes may contain amino acid residues that directly participate in binding and other amino acid residues that do not directly participate in binding. Antibody-bound epitopes can be determined using known techniques for epitope identification, such as, for example, testing the binding of an antibody to TREM1, its variants or fragments thereof, any one of the IL-1 family, IL-6 family, IL-12 family, and IL-23 family, its variants or fragments thereof, or combinations thereof.

[0042] As used herein, the term “complementarity-determining region” (CDR, i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues of an antibody variable domain whose presence is essential for antigen binding. Each variable domain typically has three CDR regions identified as CDR1, CDR2, and CDR3. The CDRs of the variable heavy chain can be CDR-H1, CDR-H2, and CDR-H3. The CDRs of the variable light chain can be CDR-L1, CDR-L2, and CDR-L3. Exemplary hypervariable rings appear 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, amino acid residues 50-56 of L2, amino acid residues 89-97 of L3, amino acid residues 31-35 of H1, amino acid residues 50-65 of H2, and amino acid residues 95-102 of H3 (Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition (1991)). Therefore, HV can be included within the corresponding CDR, and unless otherwise indicated, references to the “hypervariable loop” in the VH and VL domains should be interpreted as including the corresponding CDR, and vice versa. More highly conserved regions within the variable domains are called frame 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), primarily employing a [β]-sheet configuration linked by three hypervariable loops. The hypervariable loops in each chain are held together very closely by the FRs and, together with the hypervariable loops from the other chain, contribute to the formation of the antibody's antigen-binding site. Structural analysis of the antibodies has revealed a sequence-shape relationship between the 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 fraction of the main chain conformation, termed the "typical structure." These conformations are primarily determined by the loop length and secondarily by the presence of key residues at certain locations within the loops and frame regions, which determine the conformation through their stacking, hydrogen bonding, or ability to adopt a unique main chain conformation.The antibodies or antigen-binding fragments of this disclosure may include a CDR3 region 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 in length. The antibodies or antigen-binding fragments of this disclosure may include a CDR3 region of at least about 18 amino acids in length.

[0043] As used herein, the term “variable region” when referring to antibody use refers to the variable region of the antibody light chain alone or in combination. The variable regions of both the heavy and light chains are each composed of four frame regions (FRs) linked by three complementarity-determining regions (CDRs) (also known as hypervariable regions). The CDRs in each chain are held together very closely by the FRs and, together with the CDRs from the other chain, contribute to the formation of the antibody’s antigen-binding site. At least two techniques exist for determining CDRs: (1) methods based on cross-species sequence variability (i.e., Kabat et al., Sequences of Proteins of Immunological Interest, (5th edition, 1991, National Institutes of Health, Bethesda Md.)); and (2) methods based on the crystallographic study of antigen-antibody complexes (Al-Iazikani et al. (1997) J. Molec. Biol. 273:927-948)). A CDR can refer to a CDR defined by either method or a combination of both. Six hypervariable rings (three from the heavy chain and three from the light chain) contribute amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of the Fv containing only three antigen-specific CDRs) has the ability to recognize and bind antigens, although with lower affinity than the entire binding site.

[0044] As used herein, the term "constant region" when referring to antibody use means the constant region of the antibody light chain (i.e., the light chain constant region) alone or in combination (i.e., the heavy chain constant region). The constant region does not change relative to antigen specificity.

[0045] As used herein, the term "heavy chain region" comprises an amino acid sequence derived from a constant domain of the immunoglobulin heavy chain. A polypeptide comprising a heavy chain region comprises at least one of the following: a CH1 domain, a hinge (e.g., an upper hinge, middle hinge, and / or lower hinge) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. In one embodiment, an antibody or its antigen-binding fragment may comprise an Fc region of the immunoglobulin heavy chain (e.g., a hinge portion, a CH2 domain, and a CH3 domain). In another embodiment, the antibody or its antigen-binding fragment lacks at least one region of a constant domain (e.g., all or part of the CH2 domain). In some embodiments, at least one, and preferably all, of the constant domains are derived from the human immunoglobulin heavy chain. For example, in a preferred embodiment, the heavy chain region comprises a completely human hinge domain. In other preferred embodiments, the heavy chain region comprises a completely human Fc region (e.g., a hinge, CH2, and CH3 domain sequence from a 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" refers to the region of a heavy chain molecule that connects the CH1 and CH2 domains. The hinge region can contain approximately 25 residues and is flexible, thereby allowing the two N-terminal antigen-binding regions to move independently. The hinge region can be subdivided into three distinct domains: the upper hinge domain, the middle hinge domain, and the lower hinge domain (Roux et al. J. Immunol. 1998 161:4083).

[0047] As used herein, the term "Fv" refers to the smallest antibody fragment containing both a complete antigen recognition site and a complete antigen binding site. This fragment consists of a dimer of a tightly non-covalently associated heavy chain variable region domain and a light chain variable region domain. Folding of these two domains produces six hypervariable rings (three from the H chain and three from the L chain), which contribute amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific CDRs) can recognize and bind antigens, albeit with lower affinity than the complete binding site.

[0048] As used herein, the term “heavy chain variable region” or “VH” when referring to an antibody refers to a segment of the heavy chain containing three CDRs inserted between flanking stretches called framework regions. These framework regions are typically more conserved than the CDRs and form a scaffold supporting the CDRs.

[0049] As used herein, the term “light chain variable region” or “VL” when referring to an antibody refers to a segment of the light chain containing three CDRs inserted between flanking stretches called framework regions. These framework regions are typically more conserved than the CDRs and form a scaffold supporting the CDRs.

[0050] As used herein, the term “framework residue” or “FR” refers to those variable domain amino acid residues other than those in the hypervariable region.

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

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

[0053] As used herein, the terms "specific binding" or "preferred binding" refer to the binding of an antibody or its antigen-binding fragment to a target with a greater affinity and / or affinity than it binds 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 affinity. Methods for determining such specific binding are 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 commonly and specifically bind to two or more different epitopes. The 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, a “recombinant antibody” is an antibody comprising amino acid sequences derived from two different species or two different sources, and includes synthetic and / or non-naturally occurring molecules. By way of non-limiting example, a recombinant antibody may be an antibody comprising a non-human CDR and a human variable region framework or constant region or Fc region, an antibody having a binding domain from two different monoclonal antibodies, or an antibody comprising one or more amino acid residue mutations to increase or decrease biological activity or binding of a portion of the antibody. In some embodiments, the recombinant antibody is produced from or synthesized from a recombinant DNA molecule. In some embodiments, the antibody described herein is encoded by one or more polypeptides of one or more polynucleotides.

[0056] As used herein, “recognition,” “binding,” or “selectivity” refers to the association or binding between an antigen-binding domain and an antigen. As used herein, “antigen” refers to an antigenic substance capable of triggering an immune response in a host. Antigenic substances can be molecules, such as co-stimulatory molecules capable of triggering an immune response in a host.

[0057] As used in this article, "antibody construct" refers to a construct that may contain an antigen-binding domain and an Fc domain.

[0058] As used in this article, "binding domain" refers to either an antibody or a non-antibody domain.

[0059] As used herein, "antigen-binding domain" refers to a binding domain, whether derived from an antibody or a non-antibody, capable of binding to an antigen. When a particular conjugate or antibody construct contains more than one antigen-binding domain, these domains can be numbered (e.g., first antigen-binding domain, second antigen-binding domain, third antigen-binding domain, etc.). Different antigen-binding domains in the same conjugate or construct can target the same antigen or different antigens.

[0060] As used in this article, "antibody-antigen binding domain" refers to a binding domain derived from an antibody that can bind to an antigen.

[0061] As used herein, "Fc domain" refers to an Fc domain derived from an antibody or a non-antibody that is capable of binding to the Fc receptor. As used herein, "Fc domain" and "Fc-containing domain" are used interchangeably.

[0062] As used in this article, "target-binding domain" refers to a construct that contains an antigen-binding domain derived from an antibody or a non-antibody and is capable of binding to an antigen.

[0063] As used herein, the abbreviations for natural 1-enantiomer amino acids are conventional 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); glycine (G, Gly); histidine (H, His); isoleucine (I, Ile); 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 indicate any amino acid.

[0064] The term “pharmaceutically acceptable” is used in this article to refer to compounds, materials, compositions and / or dosage forms that, to the extent of reasonable medical judgment, are suitable for contact with the tissues of subjects, such as humans and animals, without excessive toxicity, irritation, allergic reactions or other problems or complications, and in proportion to a reasonable benefit / risk ratio.

[0065] As used herein, “pharmaceuticalally acceptable excipient” or “pharmaceuticalally acceptable carrier” means a pharmaceutically acceptable substance, composition, or medium, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating substance. Each carrier must be “acceptable” in the sense that it is compatible with other components of the formulation and is not harmful to the patient. Some examples of materials that can be used as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; (4) powdered tragacanth gum; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and… Soybean oil; (10) diols, such as propylene glycol; (11) polyols, such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) phosphate buffer solution; and (21) other non-toxic compatible substances used in pharmaceutical preparations.

[0066] Antigens can trigger an immune response. Antigens can be proteins, polysaccharides, lipids, or glycolipids that can be recognized by immune cells such as T cells or B cells. Exposing immune cells to one or more of these antigens can trigger a rapid cell division and differentiation response, resulting in the formation of clones of 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] An "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 a foreign antigen or an infectious disease antigen.

[0068] The terms “fragment of antibody,” “antibody fragment,” “functional fragment of antibody,” “antigen-binding domain,” or their grammatical equivalents are used interchangeably herein to refer to one or more fragments or portions of an antibody that retain the ability to bind specifically to an antigen (see generally Holliger et al., Nat. Biotech., 23(9):1126-1129 (2005)). Antibody fragments are intended to contain, for example, one or more CDRs, variable regions (or portions thereof), constant regions (or portions thereof), or combinations 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 a disulfide bridge at a stalk region; (iii) Fv fragments consisting of the VL and VH domains of an antibody single arm; and (iv) single-chain Fv (scFv), which is a monovalent molecule consisting of two domains (i.e., VL and VH) of an Fv fragment linked by a synthetic linker that allows the two domains to be synthesized as a single polypeptide chain (see, for example, 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 (1998)); and (v) a biantibody, said biantibody being a dimer of polypeptide chains wherein each polypeptide chain may contain a VH linked to a VL via a peptide linker, the peptide linker being too short to allow pairing between VH and VL on the same polypeptide chain, thereby driving pairing between complementary domains on different VH-VL polypeptide chains to produce a dimer molecule having two functional antigen-binding sites. Antibody fragments are known in the art and are described in more detail, for example, in 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) containing one constant domain and one variable domain for each heavy and light chain, but in which the heavy chain is truncated, resulting in the absence of the CH2 and CH3 domains (i.e., VH, CH1, VL, and CL), and may also be missing some or all of the hinge region. Fab can be generated by digesting the entire antibody with papain. Fab can refer to the region alone or in the context of a full-length antibody, an immunoglobulin construct, or a Fab fusion protein. Fab can be obtained by treating the entire antibody with pepsin and then reducing it to produce a molecule consisting of an intact light chain and a heavy chain portion containing VH and a single constant domain. Each antibody treated in this way yields two Fab fragments.

[0070] As used herein, the term "scFv" refers to an antibody fragment containing both the VH and VL domains of an antibody, wherein 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. Typically, Fv polypeptides also contain a polypeptide linker between the VH and VL domains, which allows the scFv to form the structure required for antigen binding. For a review of scFv, see Pluckthun (1994), *The Pharmacology of Monoclonal Antibodies*, Vol. 113, eds. Rosenburg and Moore (Springer-Verlag, New York), pp. 269–315. The VH and VL domain complex of an Fv fragment can also be stabilized by disulfide bonds (U.S. Patent 5,747,654).

[0071] The term "conserved amino acid substitution" or "conserved mutation" refers to the substitution of one amino acid for another amino acid with a shared characteristic. One functional approach to defining the shared characteristic between individual amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins in homologous organisms (Schulz, GE and Schirmer, RH, Principles of Protein Structure, Springer-Verlag, New York (1979)). Based on such analysis, groups of amino acids can be defined, where amino acids within a group preferentially exchange with each other and are therefore most similar to each other in their effect on the overall protein structure. Examples of conserved mutations include amino acid substitutions in the above subgroups, for example, lysine replacing arginine, and vice versa, to maintain a positive charge; glutamic acid replacing aspartic acid, and vice versa, to maintain a negative charge; serine replacing threonine, to maintain a free -OH group; and glutamine replacing asparagine, to maintain a free -NH2 group. Optionally or additionally, a therapeutic agent may comprise the amino acid sequence of a reference protein having at least one non-conserved amino acid substitution.

[0072] The term "non-conservative mutation" or "non-conservative amino acid substitution" includes amino acid substitutions between different groups, such as lysine replacing tryptophan or phenylalanine replacing 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. Non-conservative amino acid substitution can enhance the biological activity of the therapeutic agent, resulting in increased biological activity compared to the wild-type therapeutic agent.

[0073] A "multispecific antibody" is an antibody that can simultaneously bind to at least two targets with different structures (e.g., 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 having the same or different structures. Valence indicates how many binding arms or sites an antibody has for a single antigen or epitope; i.e., monovalent, bivalent, trivalent, or multivalent. The multivalent nature of an antibody means that it can utilize multiple interactions with the antigen, thereby increasing its affinity for the antigen. Specificity indicates how many antigens or epitopes an antibody can bind; i.e., 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. Multispecific, multivalent antibodies are constructs having more than one binding region with different specificities. For example, the bispecific antibody constructs disclosed herein have a first antigen-binding region and a second antigen-binding region, wherein the first antigen-binding region and the second antigen-binding region are different.

[0074] A bispecific antibody is an antibody that can simultaneously bind to two targets with different structures. Bispecific antibodies (BsAbs) and bispecific antibody fragments (bsFabs) can have at least one arm (or binding domain) that specifically binds to, for example, 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 can be an antigen produced by or associated with diseased cells, tissues, organs, or pathogens. A variety of bispecific antibodies can be generated using molecular engineering.

[0075] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid linked to it. This term includes vectors as self-replicating nucleic acid structures as well as vectors incorporated into the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0076] As used herein, the terms “host cell,” “host cell line,” and “host cell culture” are interchangeable and refer to cells in which exogenous nucleic acids have been introduced, as well as the progeny of such cells. Host cells include “transformers” (or “transformed cells”) and “transfectants” (or “transfected cells”), each comprising primary transformed or transfected cells and their derived progeny. Such progeny may not be identical to the parent cells in their nucleic acid contents and may contain mutations.

[0077] The bispecific antibody construct or composition described herein is referred to as being administered at a “therapeuticly effective amount” if the amount administered is physiologically significant. An agent is physiologically significant if its presence causes a detectable change in the physiology of the recipient subject. In a particular embodiment, a bispecific antibody construct disclosed herein is physiologically significant if its presence induces a response to an infectious or autoimmune disease state or alleviates its signs and symptoms. A physiologically significant effect can also be the induction of humoral and / or cellular immune responses in the recipient subject.

[0078] The term "linker" is used to refer to a polypeptide comprising two or more amino acid residues linked by peptide bonds and used to link one or more antigen-binding moieties or variable domains. Such linker peptides 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 comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence of SEQ ID NO: 44. In some embodiments, the linker peptide comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence of SEQ ID NO: 45.

[0079] An “Fv” or “Fv fragment” can consist solely of a single arm of an immunoglobulin, comprising a light chain variable domain (VL) and a heavy chain variable domain (VH). Therefore, an “Fv” is the smallest antibody fragment containing a complete antigen recognition and binding site. A “double-stranded” Fv fragment consists of a dimer of a tightly non-covalently associated heavy chain variable domain and a light chain variable domain. Single-stranded Fv material (scFv) can include the VH and VL domains of an immunoglobulin, where these domains are present in a single polypeptide chain and are covalently linked to each other via a linker peptide. Typically, in scFv fragments, the light and heavy chain variable domains associate in a dimer structure similar to that in double-stranded Fv material. In a single-chain Fv fragment, the variable domain of the light chain can be positioned at the N-terminus of a single polypeptide chain, followed by a linker, and the variable domain of the heavy chain can be positioned at the C-terminus of the polypeptide chain, or vice versa, the variable domain of the heavy chain can be positioned at the N-terminus, and the variable domain of the light chain at the C-terminus, with the linker peptide positioned in between. The linker peptide can be any flexible linker known in the art, for example, made of glycine and serine residues. It is also possible to further stabilize the domain association between the VH and VL domains by introducing disulfide bonds into conserved framework regions (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 called disulfide-stabilized scFv fragments (ds-scFv).

[0080] As used herein, the term “treating” (and its variations, such as “treat” or “treatment”) refers to a clinical intervention that attempts to alter the natural course of a disease or condition in a subject with a corresponding need. Treatment may be performed during the clinicopathological process. Expected effects of treatment include cure (if applicable), delay of disease onset, reduction of disease severity, relief or alleviation of one or more symptoms of the disease, improvement of the disease, reduction or improvement of any associated symptoms of the disease, or intervention against the tendency of the disease to progress.

[0081] As used herein, the term “sufficient amount” means an amount sufficient to produce the desired effect, such as an amount sufficient to modulate the immune response of a subject.

[0082] As used in this article, the terms “modulate” and “modulation” refer to reducing or suppressing, or optionally activating or increasing, an enumerated variable.

[0083] Functional antibody fragments A functional antibody fragment binds to one or more target proteins. In some embodiments, the functional antibody fragment promotes the degradation of one or more target proteins. In some embodiments, the functional antibody fragment induces the degradation of one or more target proteins. In some embodiments, the functional antibody fragment induces / promotes the cleavage of one or more target proteins. In some embodiments, the functional antibody fragment induces / promotes the internalization of one or more target proteins. In some embodiments, the functional antibody fragment induces / promotes the shedding of one or more target proteins. In some embodiments, the functional antibody fragment induces / promotes the downregulation of the expression of one or more target proteins. In some embodiments, the functional antibody fragment blocks the activity of one or more downstream signaling proteins mediated by one or more target proteins. In some embodiments, the functional antibody fragment blocks the expression of one or more downstream signaling proteins mediated by one or more target proteins. In some embodiments, the target proteins are directly and / or indirectly associated with inflammation. Therefore, in some embodiments, administration of the functional antibody fragment to a subject may result in a reduction in inflammation compared to a subject who has not been administered the functional antibody fragment. In some implementations, the target protein includes TREM1, any of the IL-1 family proteins, any of the IL-6 family proteins, any of the IL-12 family proteins and any of the IL-23 family proteins, their variants, their functional fragments or combinations thereof.

[0084] Functional antibody fragments that recognize specific epitopes can be generated using known techniques. Functional antibody fragments are the antigen-binding portions of antibodies, such as F(ab')2, Fab', F(ab)2, Fab, Fv, scFv, etc. F(ab')2 fragments can be generated by digesting antibody molecules with pepsin, and Fab' fragments can be generated by reducing the disulfide bridges of F(ab')2 fragments. Optionally, Fab' expression libraries can be constructed (Huse et al., 1989, Science, 246:1274-1281) to allow for rapid and easy identification of monoclonal Fab' fragments with desired specificity. F(ab)2 fragments can be generated by digesting antibodies with papain.

[0085] Single-chain Fv molecules (scFv) may contain VL and VH domains. The VL and VH domains associate to form a target binding site. These two domains may also be covalently linked via a peptide linker (L). Methods for preparing scFv molecules and designing suitable peptide linkers are described in U.S. Patent Nos. 4,704,692, 4,946,778, Raag and Whitlow, FASEB 9:73-80 (1995), and Bird and Walker, TIBTECH 9:132-137 (1991).

[0086] Techniques for generating single-domain antibodies (DABs or VHHs) are also known in the art, such as those disclosed, for example, by Cossins et al. (2006, Prot Express Purif 51:253-259), which are incorporated herein by reference. Single-domain antibodies can be obtained, for example, from camels, alpacas, or llamas using standard immunization techniques. (See, for example, 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 can 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 only about 50% camel heavy chain IgG antibodies (HCAbs) (Maass et al., 2007). Alpacas can be immunized with known antigens such as TNF-α, and VHHs that bind to and neutralize target antigens can be isolated (Maass et al., 2007). PCR primers for amplifying almost all alpaca VHH coding sequences have been identified and can be used to construct alpaca VHH phage display libraries, which can be used for antibody fragment isolation using standard biopanning techniques well known in the art (Maass et al., 2007). In some embodiments, VHH antibody fragments can be used in claimed compositions and methods.

[0087] Antibody fragments can be generated by proteolysis of full-length antibodies or by the reaction of E. coli (…). E. coliAntibody fragments can be prepared by expressing DNA encoding the fragment in another host. Antibody fragments can be obtained by digesting full-length antibodies with pepsin or papain using conventional methods. These methods are described, for example, by Golden, U.S. Patents 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., METHODS INENZYMOLOGY VOL. 1, p. 422 (Academic Press 1967); and Coligan, pp. 2.8.1–2.8.10 and 2.10.–2.10.4.

[0088] Engineered protein constructs This document discloses engineered protein constructs. In some embodiments, the engineered protein constructs described herein comprise a TREM1 binding moiety, an IL-1 binding moiety, an IL-6 binding moiety, an IL-12 binding moiety, an IL-23 binding moiety, or a combination thereof. In some embodiments, the engineered protein constructs described herein comprise the multispecific molecule described herein. A multispecific molecule is an antibody capable of binding to at least two different targets. In some embodiments, the multispecific molecule targets at least two epitopes selected from TREM1, proteins selected from the IL-1 family, proteins selected from the IL-6 family, proteins selected from the IL-12 family, proteins selected from the IL-23 family, their variants, or functional fragments thereof. In some embodiments, the at least two different targets comprise two different epitopes. In some embodiments, the two different epitopes are: (a) TREM1, its variants, or functional fragments thereof, and (b) a protein selected from any one of the IL-1 family, IL-6 family, IL-12 family, and IL-23 family, its variants, or functional fragments thereof. Therefore, in some embodiments, the engineered protein constructs described herein are capable of binding at least two different epitopes, wherein the at least two different epitopes are: (a) TREM1, its variants or functional fragments thereof, and (b) a protein, its variants or functional fragments thereof selected from any one of the IL-1 family, IL-6 family, IL-12 family and IL-23 family.

[0089] Methods for preparing multispecific antibodies are known in the art. Traditionally, the recombinant generation of multispecific antibodies is based on the co-expression of two immunoglobulin heavy / light chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983)). Purification of the correct molecule is typically accomplished by an affinity chromatography step. Similar procedures are disclosed in WO 93 / 08829 and Traunecker et al., EMBO J., 10:3655-3659 (1991).

[0090] Antibodies fall into five main classes: IgA, IgD, IgE, IgG, and IgM, and several of these can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are designated α, δ, ε, γ, and µ, respectively. Therefore, the multispecific antibodies described herein encompass κ constant regions, λ constant regions, α constant regions, γ constant regions, δ constant regions, ε constant regions, μ constant regions, their functional fragments, or combinations thereof.

[0091] The class of an antibody or immunoglobulin refers to the type of constant domain or constant region possessed by 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 comprise a light chain. In some embodiments, the light chain comprises a κ light chain or a λ light chain. Multispecific antibodies such as κ antibodies or λ antibodies can be prepared using any of a variety of techniques recognized in the art, including those disclosed in WO 2012 / 023053, the contents of which are incorporated herein by reference in their entirety.

[0094] In some embodiments, antibody variable domains having desired binding specificity (antibody-antigen combining sites) can be linked to immunoglobulin constant domain sequences to form multispecific antibodies. In some embodiments, the fusion containing the antibody variable domain is preferably linked to an immunoglobulin heavy chain constant domain, wherein the immunoglobulin heavy chain constant domain includes at least a portion of a hinge region, a CH2 region, and a CH3 region. In some embodiments, it is preferred that at least one fusion contains a first heavy chain constant region (CH1) containing a site essential for light chain binding. DNA encoding the immunoglobulin heavy chain fusion and (if desired) the immunoglobulin light chain can be inserted into separate expression vectors and can be 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 a pair of antibody molecules described herein is engineered to maximize the percentage of heterodimers that can be recovered from recombinant cell cultures. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains to form protrusions or knots (e.g., tyrosine or tryptophan). By replacing the large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine), compensating cavities or holes of the same or similar size as the one or more large side chains are created at the interface of the second antibody molecule. This provides a mechanism to increase the yield of heterodimers rather than other unwanted end products such as homodimers.

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

[0097] Various techniques for preparing and isolating functional bispecific antibody fragments directly from recombinant cell cultures are also described. For example, leucine zippers have been used to generate bispecific antibodies. Kostelny et al., J. Immunol. 148(5):1547-1553 (1992). Leucine zipper peptides from Fos and Jun proteins are linked to the Fab' portion of two different antibodies via gene fusion. Antibody homodimers are reduced in the hinge region to form monomers and then oxidized to form antibody heterodimers. This method can also be used to generate antibody homodimers. The “biantibody” technique described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) provides an alternative mechanism for preparing functional bispecific antibody fragments. The functional fragments contain a heavy chain variable domain (VH) linked to a light chain variable domain (VL) via a linker that is too short to allow pairing between the two domains on the same chain. Therefore, 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 preparing functional 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 more than two valences are envisioned. For example, trispecific 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 a protein antigen of the present invention. Optionally, the anti-antigen arm of an immunoglobulin molecule can be combined with an arm that binds to triggering molecules on leukocytes such as T cell receptor molecules (e.g., CD2, CD3, CD28, or B7) or Fc receptors (FcγRs) of IgG such as FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16) to focus cellular defense mechanisms on cells expressing a specific antigen. Bispecific antibodies can also be used to direct cytotoxic agents to cells expressing a specific antigen. These antibodies can have an antigen-binding arm and an arm that binds to cytotoxic agents or radionuclide chelators such as EOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody of interest binds to the protein antigens described herein and further binds to tissue factor (TF).

[0099] Several strategies have been used to generate the multispecific molecules described herein (e.g., bispecific and trispecific molecules), such as chemical cross-linking of functional antibody fragments, forced heterodimerization, tetradox hybridoma technology, fusion of functional antibody fragments via peptide linkers, and the use of single-domain antibodies. The availability of recombinant DNA technology has led to the generation of various bispecific antibody formats (see, for example, Ridgway JB et al. (1996) Protein Eng9: 617-621). Linkers and mutations are frequently introduced into different regions of the antibody to force heterodimer formation or to link different binding moieties into a single molecule.

[0100] IL-1 family As described herein, the IL-1 family includes interleukin-1α cytokine (IL-1α), interleukin-1β cytokine (IL-1β), interleukin-1 receptor antagonist (IL1RN), interleukin-18 (IL-18), interleukin-36α (IL-36α), interleukin-36β (IL-36β), interleukin-36ϒ (IL-36ϒ), interleukin-36 receptor antagonist (IL36RN), interleukin-37 (IL-37), interleukin-38 (IL-38), fragments thereof, variants thereof, multimeric forms thereof, or combinations thereof. In some embodiments, IL-1 family proteins interact with interleukin-1 receptor-like 1 (IL1RL1), interleukin-1 receptor-like 2 (IL1RL2), interleukin-1 receptor accessory protein (IL1RAP), or combinations thereof. The amino acid sequences of IL-1α, IL-1β, IL-18, IL-36α, IL-36β, IL-36γ, IL36RN, IL-37, IL-38, IL1RL1, IL1RL2 and IL1RAP are listed in Table 1.

[0101] Table 1. Amino acid sequences of IL-1 family proteins

[0102] In some embodiments, the multispecific antibody described herein binds to at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the amino acid sequence listed in Table 1. In some embodiments, the multispecific molecule described herein binds to at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the amino acid sequence of any one of SEQ ID NOs: 1-5 and 399-407. In some embodiments, the multispecific molecule described herein may bind to: (a) an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the amino acid sequence of any one of SEQ ID NOs: 1-5, 400-404, and 406-407; and (b) TREM1, its functional fragments, its variants, or combinations thereof. In some embodiments, the multispecific molecules described herein can be bound to: (a) an amino acid sequence that is at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to any one of SEQ ID NO:1-5 and 400-401; and (b) TREM1, its functional fragments, its variants, or combinations thereof.

[0103] In some embodiments, the multispecific molecules described herein comprise at least one of the CDR-Hs or variants thereof described in Table 2, wherein the multispecific molecules are capable of binding at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NOs: 1-5 and 399-407. In some embodiments, the multispecific molecules described herein comprise any one of the CDR-H1s or variants thereof described in Table 2, any one of the CDR-H2s or variants thereof described in Table 2, and any one of the CDR-H3s or variants thereof described in Table 2, wherein the multispecific molecules are capable of binding at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NOs: 1-5 and 399-407.

[0104] In some embodiments, the multispecific molecules described herein comprise any combination of CDR-Hs or variants thereof described in Table 2, wherein the multispecific molecules are capable of binding at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NOs: 1-5 and 399-407. In some embodiments, the multispecific molecules described herein comprise at least one of the CDR-Hs or variants thereof described in Table 2, wherein the multispecific molecules are capable of binding at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NOs: 1-5, 400-404, and 406-407. In some embodiments, the CDR-H variants comprise at least one, at least two, or at least three substitutions, deletions, additions, or combinations thereof relative to the corresponding parental CDR-H sequences described in Table 2. In some embodiments, the CDR-H or its variants contain 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% of the same amino acid sequence as the corresponding parental CDR-H sequence described in Table 2.

[0105] In some embodiments, the multispecific molecules described herein comprise an IL-1 binding domain and a TREM1 binding domain, wherein (a) the IL-1 binding domain comprises any one of the combinations of CDR-H1, CDR-H2, and CDR-H3 described in Table 2, (b) the IL-1 binding domain can bind at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NO: 1-5 and 400-401, and (c) the TREM1 binding domain can bind TREM1, its functional fragments, its variants, or combinations thereof.

[0106] In some embodiments, the engineered protein constructs described herein include an IL-1 binding moiety, wherein (a) the IL-1 binding moiety comprises any one of the combinations of CDR-H1, CDR-H2 and CDR-H3 described in Table 2, and (b) the IL-1 binding moiety can bind at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% of the same amino acid sequence as any one of SEQ ID NO: 1-5 and 400-401. In some embodiments, the engineered protein constructs described herein include an IL-1 binding region and a TREM1 binding region, wherein (a) the IL-1 binding region includes any one of the combinations of CDR-H1, CDR-H2, and CDR-H3 described in Table 2, (b) the IL-1 binding region can bind at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NO: 1-5 and 400-401, and (c) the TREM1 binding region can bind TREM1, its functional fragments, its variants, or combinations thereof.

[0107] Table 2. Combinations of CDR-H for binding to IL-1 family proteins

[0108] In some embodiments, the multispecific molecules described herein comprise 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% of the same amino acid sequence as any one of the VH sequences described in Table 3, wherein the multispecific molecules are capable of binding to at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NO: 1-5 and 399-407.

[0109] In some embodiments, the multispecific molecule described herein comprises at least one VH sequence, wherein the multispecific molecule is capable of binding at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NOs: 1-5, 400-404, and 406-407. In some embodiments, the multispecific molecule described herein comprises 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% of the same amino acid sequence as any one of the VH sequences described in Table 3, wherein the multispecific molecule is capable of binding at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NOs: 1-5 and 400-401. In some embodiments, the multispecific molecules described herein comprise an IL-1 binding domain and a TREM1 binding domain, wherein (a) the IL-1 binding domain comprises 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% of the same amino acid sequence as any one of the VH sequences described in Table 3, (b) the IL-1 binding domain can bind at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NO: 1-5 and 400-401, and (c) the TREM1 binding domain can bind TREM1, its functional fragments, its variants, or combinations thereof.

[0110] In some embodiments, the engineered protein constructs described herein include an IL-1 binding moiety, wherein (a) the IL-1 binding moiety contains 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% of the same amino acid sequence as any one of the VH sequences described in Table 3, and (b) the IL-1 binding moiety can bind to at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NO: 1-5 and 400-401. In some embodiments, the engineered protein constructs described herein include an IL-1 binding region and a TREM1 binding region, wherein (a) the IL-1 binding region contains 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% of the same amino acid sequence as any one of the VH sequences described in Table 3; (b) the IL-1 binding region can bind at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NO: 1-5 and 400-401; and (c) the TREM1 binding region can bind TREM1, its functional fragments, its variants, or combinations thereof.

[0111] Table 3. Exemplary VH sequences for binding to IL-1 family proteins

[0112] In some embodiments, the multispecific molecule described herein comprises at least one heavy chain (HC) region, wherein the multispecific molecule can bind at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NOs: 1-5, 400-404, and 406-407. In some embodiments, the multispecific molecule described herein comprises an IL-1 binding region, wherein the IL-1 binding region comprises an HC region. In some embodiments, the HC region of the IL-1 binding region comprises 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% of the same VH sequence as any one of the amino acid sequences described in Table 3.

[0113] In some implementations, the HC region of the IL-1 binding region contains the VH sequence and the sequence ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 481) 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% of the same amino acid sequence, wherein the C-terminus of the VH sequence is connected to the N-terminus of the amino acid sequence.

[0114] In some implementations, the HC region of the IL-1 binding region contains the VH sequence and the sequence ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 482) 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% of the same amino acid sequence, wherein the C-terminus of the VH sequence is connected to the N-terminus of the amino acid sequence.

[0115] In some embodiments, the multispecific molecules described herein comprise at least one of the CDR-Ls or variants thereof described in Table 4, wherein the multispecific molecules are capable of binding at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NOs: 1-5 and 399-407. In some embodiments, the multispecific molecules described herein comprise any one of the CDR-L1s or variants thereof described in Table 4, any one of the CDR-L2s or variants thereof described in Table 4, and any one of the CDR-L3s or variants thereof described in Table 4, wherein the multispecific molecules are capable of binding at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NOs: 1-5 and 399-407.

[0116] In some embodiments, the multispecific molecules described herein comprise any combination of the CDR-Ls or variants thereof described in Table 4, wherein the multispecific molecules are capable of binding at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NOs: 1-5 and 399-407. In some embodiments, the multispecific molecules described herein comprise at least one of the CDR-Ls or variants thereof described in Table 4, wherein the multispecific molecules can bind at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NOs: 1-5, 400-404, and 406-407. In some embodiments, the CDR-L variants comprise at least one, at least two, or at least three substitutions, deletions, additions, or combinations thereof relative to the corresponding parental CDR-L sequences described in Table 4. In some embodiments, the CDR-L or its variants contain 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% of the same amino acid sequence as the corresponding parental CDR-L sequence described in Table 4.

[0117] In some embodiments, the engineered protein constructs described herein include an IL-1 binding moiety, wherein (a) the IL-1 binding moiety comprises any one of the combinations of CDR-L1, CDR-L2 and CDR-L3 described in Table 4, and (b) the IL-1 binding moiety can bind at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% of the same amino acid sequence as any one of SEQ ID NO: 1-5 and 400-401. In some embodiments, the engineered protein constructs described herein include an IL-1 binding region and a TREM1 binding region, wherein (a) the IL-1 binding region includes any one of the combinations of CDR-L1, CDR-L2, and CDR-L3 described in Table 4, (b) the IL-1 binding region can bind at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NOs: 1-5 and 400-401, and (c) the TREM1 binding region can bind TREM1, its functional fragments, its variants, or combinations thereof. In some embodiments, the multispecific molecule described herein comprises an IL-1 binding domain and a TREM1 binding domain, wherein (a) the IL-1 binding domain comprises any one of the combinations of CDR-L1, CDR-L2 and CDR-L3 described in Table 4, (b) the IL-1 binding domain can bind at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% of the same amino acid sequence as any one of SEQ ID NO: 1-5 and 400-401, and (c) the TREM1 binding domain can bind TREM1, its functional fragments, its variants or combinations thereof.

[0118] Table 4. Combinations of CDR-Ls for binding to IL-1 family proteins

[0119] In some embodiments, the multispecific molecules described herein comprise combinations of CDRs, wherein the CDRs comprise 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 wherein the combination is any one of the combinations provided in Table 5.

[0120] Table 5. Exemplary CDR combinations for antibodies targeting IL-1 family proteins

[0121] In some embodiments, the multispecific molecules described herein comprise 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% of the same amino acid sequence as any one of the VL sequences described in Table 6, wherein the multispecific molecules are capable of binding to at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NO: 1-5 and 399-407.

[0122] In some embodiments, the multispecific molecule described herein comprises at least one VL sequence, wherein the multispecific molecule is capable of binding at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NO: 1-5, 400-404, and 406-407. In some embodiments, the multispecific molecule described herein comprises 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% of the same amino acid sequence as any one of the VL sequences described in Table 6, wherein the multispecific molecule is capable of binding at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NO: 1-5 and 400-401. In some embodiments, the engineered protein constructs described herein include an IL-1 binding moiety, wherein (a) the IL-1 binding moiety contains 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% of the same amino acid sequence as any one of the VL sequences described in Table 6, and (b) the IL-1 binding moiety can bind to at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NO:1-5 and 400-401. In some embodiments, the engineered protein constructs described herein include an IL-1 binding region and a TREM1 binding region, wherein (a) the IL-1 binding region contains 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% of the same amino acid sequence as any one of the VL sequences described in Table 6; (b) the IL-1 binding region can bind at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NO: 1-5 and 400-401; and (c) the TREM1 binding region can bind TREM1, its functional fragments, its variants, or combinations thereof.In some embodiments, the multispecific molecules described herein comprise an IL-1 binding domain and a TREM1 binding domain, wherein (a) the IL-1 binding domain comprises 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% of the same amino acid sequence as any one of the VL sequences described in Table 6; (b) the IL-1 binding domain can bind at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NO: 1-5 and 400-401; and (c) the TREM1 binding domain can bind TREM1, its functional fragments, its variants, or combinations thereof.

[0123] Table 6. Exemplary VL sequences for binding to IL-1 family proteins

[0124] In some embodiments, the multispecific molecule described herein comprises at least one light chain (LC) region, wherein the multispecific molecule can bind at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NOs: 1-5, 400-404, and 406-407. In some embodiments, the multispecific molecule described herein comprises an IL-1 binding region, wherein the IL-1 binding region comprises an LC region. In some embodiments, the LC region of the IL-1 binding region comprises 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% of the same VL sequence as any one of the amino acid sequences described in Table 6.

[0125] In some embodiments, the LC region of the IL-1 binding region comprises a VL sequence and 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 RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 483), wherein the C-terminus of the VL sequence is linked to the N-terminus of the amino acid sequence.

[0126] In some embodiments, the multispecific molecule described herein comprises: (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 of the amino acid sequences described 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 of the amino acid sequences described in Table 6, wherein the multispecific molecule comprises a VH sequence and a VL sequence according to any of the combinations described in Table 7.

[0127] Table 7. Exemplary combinations of VH and VL sequences for binding to IL-1 family proteins.

[0128] IL-6 family As described herein, the IL-6 family includes interleukin-6 cytokine (IL-6), interleukin-11 cytokine (IL-11), fragments thereof, variants thereof, multimeric forms thereof, or combinations thereof. In some embodiments, IL-6 family proteins bind to the interleukin-6 receptor (IL-6R). The amino acid sequences of IL-6, IL-11, and IL-6R are listed in Table 8.

[0129] Table 8. Amino acid sequences of IL-6 family proteins

[0130] In some embodiments, the multispecific molecular binding described herein is at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to any of the sequences listed in Table 8. In some embodiments, the multispecific molecular binding described herein is at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to any of SEQ ID NO: 83-84 and 442. In some embodiments, the multispecific molecular binding described herein is: (a) an amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to any of SEQ ID NO: 83-84 and 442; and (b) TREM1, its functional fragments, its variants, or combinations thereof.

[0131] In some embodiments, the multispecific molecules described herein comprise at least one of the CDR-Hs or variants thereof described in Table 9, wherein the multispecific molecules are capable of binding at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NO: 83-84. In some embodiments, the multispecific molecules described herein comprise any one of the CDR-H1 or variants thereof described in Table 9, any one of the CDR-H2 or variants thereof described in Table 9, and any one of the CDR-H3 or variants thereof described in Table 9, wherein the multispecific molecules are capable of binding at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NO: 83-84. In some embodiments, the multispecific molecules described herein comprise any combination of CDR-H or variants thereof described in Table 9, wherein the multispecific molecules are capable of binding to at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any of SEQ ID NO: 83-84. In some embodiments, the CDR-H variants comprise at least one, at least two, or at least three substitutions, deletions, additions, or combinations thereof relative to the corresponding parental CDR-H sequence described in Table 9. In some embodiments, the CDR-H or variants thereof comprise 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% of the same amino acid sequence as the corresponding parental CDR-H sequence described in Table 9.

[0132] In some embodiments, the engineered protein constructs described herein include an IL-6 binding moiety, wherein (a) the IL-6 binding moiety comprises any one of the combinations of CDR-H1, CDR-H2 and CDR-H3 described in Table 9, and (b) the IL-6 binding moiety can bind at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% of the same amino acid sequence as any one of SEQ ID NO: 83-84. In some embodiments, the engineered protein constructs described herein include an IL-6 binding region and a TREM1 binding region, wherein (a) the IL-6 binding region includes any one of the combinations of CDR-H1, CDR-H2, and CDR-H3 described in Table 9, (b) the IL-6 binding region can bind at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NO:83-84, and (c) the TREM1 binding region can bind TREM1, its functional fragments, its variants, or combinations thereof. In some embodiments, the multispecific molecule described herein comprises an IL-6 binding domain and a TREM1 binding domain, wherein (a) the IL-6 binding domain comprises any one of the combinations of CDR-H1, CDR-H2 and CDR-H3 described in Table 9, (b) the IL-6 binding domain can bind at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% of the same amino acid sequence as any one of SEQ ID NO: 83-84, and (c) the TREM1 binding domain can bind TREM1, its functional fragments, its variants or combinations thereof.

[0133] Table 9. Combinations of CDR-H for binding to IL-6 family proteins

[0134] In some embodiments, the multispecific molecule described herein comprises at least one VH sequence, wherein the multispecific molecule is capable of binding at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of the VH sequences described in Table 10, wherein the multispecific molecule is capable of binding at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the same amino acid sequence as any one of the VH sequences described in SEQ ID NO: 83-84. In some embodiments, the engineered protein construct described herein includes an IL-6 binding moiety, wherein (a) the IL-6 binding moiety contains 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% of the same amino acid sequence as any one of the VH sequences described in Table 10, and (b) the IL-6 binding moiety can bind to at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NO: 83-84. In some embodiments, the engineered protein constructs described herein include an IL-6 binding region and a TREM1 binding region, wherein (a) the IL-6 binding region contains 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% of the same amino acid sequence as any one of the VH sequences described in Table 10; (b) the IL-6 binding region can bind at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NO: 83-84; and (c) the TREM1 binding region can bind TREM1, its functional fragments, its variants, or combinations thereof.In some embodiments, the multispecific molecule described herein comprises an IL-6 binding domain and a TREM1 binding domain, wherein (a) the IL-6 binding domain comprises 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% of the same amino acid sequence as any one of the VH sequences described in Table 10, (b) the IL-6 binding domain may bind at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NO: 83-84, and (c) the TREM1 binding domain may bind TREM1, its functional fragments, its variants, or combinations thereof.

[0135] Table 10. Exemplary VH sequences for binding to IL-6 family proteins

[0136] In some embodiments, the multispecific molecule described herein comprises at least one heavy chain (HC) region, wherein the multispecific molecule can bind at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NOs: 83-84 and 442. In some embodiments, the multispecific molecule described herein comprises an IL-6 binding region, wherein the IL-6 binding region comprises an HC region. In some embodiments, the HC region of the IL-6 binding region comprises 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% of the same VH sequence as any one of the amino acid sequences described in Table 10.

[0137] In some implementations, the HC region of the IL-6 binding region contains the VH sequence and the sequence ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 481) 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% of the same amino acid sequence, wherein the C-terminus of the VH sequence is connected to the N-terminus of the amino acid sequence.

[0138] In some implementations, the HC region of the IL-6 binding region contains the VH sequence and the sequence ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 482) 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% of the same amino acid sequence, wherein the C-terminus of the VH sequence is connected to the N-terminus of the amino acid sequence.

[0139] In some embodiments, the multispecific molecules described herein comprise at least one of the CDR-Ls or variants thereof described in Table 11, wherein the multispecific molecules are capable of binding at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NO: 83-84. In some embodiments, the multispecific molecules described herein comprise any one of the CDR-L1s or variants thereof described in Table 11, any one of the CDR-L2s or variants thereof described in Table 11, and any one of the CDR-L3s or variants thereof described in Table 11, wherein the multispecific molecules are capable of binding at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NO: 83-84. In some embodiments, the multispecific molecules described herein comprise any combination of the CDR-L or variants thereof described in Table 11, wherein the multispecific molecules are capable of binding to at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any of SEQ ID NO: 83-84. In some embodiments, the CDR-L variants comprise at least one, at least two, or at least three substitutions, deletions, additions, or combinations thereof relative to the corresponding parental CDR-L sequence described in Table 11. In some embodiments, the CDR-L or variants thereof comprise 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% of the same amino acid sequence as the corresponding parental CDR-L sequence described in Table 11.

[0140] In some embodiments, the engineered protein constructs described herein include an IL-6 binding moiety, wherein (a) the IL-6 binding moiety includes any one of the combinations of CDR-L1, CDR-L2 and CDR-L3 described in Table 11, and (b) the IL-6 binding moiety can bind at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% of the same amino acid sequence as any one of SEQ ID NO: 83-84. In some embodiments, the engineered protein constructs described herein include an IL-6 binding region and a TREM1 binding region, wherein (a) the IL-6 binding region includes any one of the combinations of CDR-L1, CDR-L2 and CDR-L3 described in Table 11, (b) the IL-6 binding region can bind at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% of the same amino acid sequence as any one of SEQ ID NO: 83-84, and (c) the TREM1 binding region can bind TREM1, its functional fragments, its variants or combinations thereof. In some embodiments, the multispecific molecule described herein comprises an IL-6 binding domain and a TREM1 binding domain, wherein (a) the IL-6 binding domain comprises any one of the combinations of CDR-L1, CDR-L2 and CDR-L3 described in Table 11, (b) the IL-6 binding domain can bind at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% of the same amino acid sequence as any one of SEQ ID NO: 83-84, and (c) the TREM1 binding domain can bind TREM1, its functional fragments, its variants or combinations thereof.

[0141] Table 11. Combinations of CDR-Ls for binding to IL-6 family proteins

[0142] In some embodiments, the multispecific molecules described herein comprise combinations of CDRs, wherein the CDRs comprise 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 wherein the combination is any one of the combinations provided in Table 12.

[0143] Table 12. Exemplary CDR combinations for antibodies targeting IL-6 family proteins

[0144] In some embodiments, the multispecific molecule described herein comprises at least one VL sequence, wherein the multispecific molecule is capable of binding at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of the VL sequences described in Table 13, wherein the multispecific molecule is capable of binding at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the same amino acid sequence as any one of the VL sequences described in SEQ ID NO: 83-84. In some embodiments, the engineered protein constructs described herein include an IL-6 binding moiety, wherein (a) the IL-6 binding moiety contains 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% of the same amino acid sequence as any one of the VL sequences described in Table 13, and (b) the IL-6 binding moiety can bind to at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NO: 83-84. In some embodiments, the engineered protein constructs described herein include an IL-6 binding region and a TREM1 binding region, wherein (a) the IL-6 binding region contains 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% of the same amino acid sequence as any one of the VL sequences described in Table 13, (b) the IL-6 binding region can bind at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NO: 83-84, and (c) the TREM1 binding region can bind TREM1, its functional fragments, its variants, or combinations thereof.In some embodiments, the multispecific molecule described herein comprises an IL-6 binding domain and a TREM1 binding domain, wherein (a) the IL-6 binding domain comprises 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% of the same amino acid sequence as any one of the VL sequences described in Table 13, (b) the IL-6 binding domain may bind at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NO: 83-84, and (c) the TREM1 binding domain may bind TREM1, its functional fragments, its variants, or combinations thereof.

[0145] Table 13. Exemplary VL sequences for binding to IL-6 family proteins

[0146] In some embodiments, the multispecific molecule described herein comprises at least one light chain (LC) region, wherein the multispecific molecule can bind at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NOs: 83-84 and 442. In some embodiments, the multispecific molecule described herein comprises an IL-6 binding region, wherein the IL-6 binding region comprises an LC region. In some embodiments, the LC region of the IL-6 binding region comprises 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% of the same VL sequence as any one of the amino acid sequences described in Table 13.

[0147] In some embodiments, the LC region of the IL-6 binding region comprises a VL sequence and 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 RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 483), wherein the C-terminus of the VL sequence is linked to the N-terminus of the amino acid sequence.

[0148] In some embodiments, the multispecific molecule described herein comprises: (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 of the amino acid sequences described in Table 10; 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 of the amino acid sequences described in Table 13, wherein the multispecific molecule comprises a VH sequence and a VL sequence according to any of the combinations described in Table 14.

[0149] Table 14. Exemplary combinations of VH and VL sequences for binding to IL-6 family proteins.

[0150] IL-12 family As described herein, the IL-12 family includes interleukin-12α cytokine (IL-12α), interleukin-12β cytokine (IL-12β), interleukin-12 receptor β1 (IL12Rβ1), interleukin-12 receptor β2 (IL12Rβ2), interleukin-23α (IL-23α), interleukin-23 receptor (IL23R), interleukin-27α (IL-27α), interleukin-27β (IL-27β), their functional fragments, their variants, their multimeric forms, or combinations thereof. The amino acid sequences of IL-12 family proteins are listed in Table 15.

[0151] Table 15. Amino acid sequences of IL-12 family proteins

[0152] In some embodiments, the multispecific molecular binding described herein is at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to any of the sequences listed in Table 15. In some embodiments, the multispecific molecular binding described herein is at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to any of SEQ ID NOs: 212-213, 234, and 443-444. In some embodiments, the multispecific molecular binding described herein is: (a) at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to any of SEQ ID NOs: 212-213, 234, and 443-444; and (b) TREM1, its functional fragments, its variants, or combinations thereof.

[0153] In some embodiments, the multispecific molecules described herein comprise at least one of the CDR-Hs or variants thereof described in Table 16, wherein the multispecific molecules are capable of binding at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NOs: 212-213 and 443. In some embodiments, the multispecific molecules described herein comprise any one of the CDR-H1s or variants thereof described in Table 16, any one of the CDR-H2s or variants thereof described in Table 16, and any one of the CDR-H3s or variants thereof described in Table 16, wherein the multispecific molecules are capable of binding at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NOs: 212-213 and 443. In some embodiments, the multispecific molecules described herein comprise any combination of CDR-H or variants thereof described in Table 16, wherein the multispecific molecules are capable of binding to at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NOs: 212-213 and 443. In some embodiments, the CDR-H variants comprise at least one, at least two, or at least three substitutions, deletions, additions, or combinations thereof relative to the corresponding parental CDR-H sequence described in Table 16. In some embodiments, the CDR-H or variants thereof comprise 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% of the same amino acid sequence as the corresponding parental CDR-H sequence described in Table 16.

[0154] In some embodiments, the engineered protein constructs described herein include an IL-12 binding moiety, wherein (a) the IL-12 binding moiety comprises any one of the combinations of CDR-H1, CDR-H2 and CDR-H3 described in Table 16, and (b) the IL-12 binding moiety can bind at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% of the same amino acid sequence as any one of SEQ ID NO: 212-213 and 443. In some embodiments, the engineered protein constructs described herein include an IL-12 binding region and a TREM1 binding region, wherein (a) the IL-12 binding region includes any one of the combinations of CDR-H1, CDR-H2, and CDR-H3 described in Table 16, (b) the IL-12 binding region can bind at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NO: 212-213 and 443, and (c) the TREM1 binding region can bind TREM1, its functional fragments, its variants, or combinations thereof. In some embodiments, the multispecific molecule described herein comprises an IL-12 binding domain and a TREM1 binding domain, wherein (a) the IL-12 binding domain comprises any one of the combinations of CDR-H1, CDR-H2, and CDR-H3 described in Table 16, (b) the IL-12 binding domain can bind at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NO: 212-213 and 443, and (c) the TREM1 binding domain can bind TREM1, its functional fragments, its variants, or combinations thereof.

[0155] Table 16. Combinations of CDR-H for binding to IL-12 family proteins

[0156] In some embodiments, the multispecific molecule described herein comprises at least one VH sequence, wherein the multispecific molecule is capable of binding at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NOs: 212-213, 234, and 443-444. In some embodiments, the multispecific molecule described herein comprises 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% of the same amino acid sequence as any one of the VH sequences described in Table 17, wherein the multispecific molecule is capable of binding at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NOs: 212-213 and 443. In some embodiments, the engineered protein constructs described herein include an IL-12 binding moiety, wherein (a) the IL-12 binding moiety contains 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% of the same amino acid sequence as any one of the VH sequences described in Table 17, and (b) the IL-12 binding moiety can bind to at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NOs: 212-213 and 443. In some embodiments, the engineered protein constructs described herein include an IL-12 binding region and a TREM1 binding region, wherein (a) the IL-12 binding region contains 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% of the same amino acid sequence as any one of the VH sequences described in Table 17; (b) the IL-12 binding region may bind at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NOs: 212-213 and 443; and (c) the TREM1 binding region may bind TREM1, its functional fragments, its variants, or combinations thereof.In some embodiments, the multispecific molecule described herein comprises an IL-12 binding domain and a TREM1 binding domain, wherein (a) the IL-12 binding domain comprises 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% of the same amino acid sequence as any one of the VH sequences described in Table 17, (b) the IL-12 binding domain may bind at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NOs: 212-213 and 443, and (c) the TREM1 binding domain may bind TREM1, its functional fragments, its variants, or combinations thereof.

[0157] Table 17. Exemplary VH sequences for binding to IL-12 family proteins

[0158] In some embodiments, the multispecific molecule described herein comprises at least one heavy chain (HC) region, wherein the multispecific molecule can bind at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NOs: 212-213, 234, and 443-444. In some embodiments, the multispecific molecule described herein comprises an IL-12 binding region, wherein the IL-12 binding region comprises an HC region. In some embodiments, the HC region of the IL-12 binding region comprises 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% of the same VH sequence as any one of the amino acid sequences described in Table 17.

[0159] In some implementations, the HC region of the IL-12 binding region contains the VH sequence and the sequence ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 481) 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% of the same amino acid sequence, wherein the C-terminus of the VH sequence is connected to the N-terminus of the amino acid sequence.

[0160] In some implementations, the HC region of the IL-12 binding region contains the VH sequence and the sequence ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 482) 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% of the same amino acid sequence, wherein the C-terminus of the VH sequence is connected to the N-terminus of the amino acid sequence.

[0161] In some embodiments, the multispecific molecules described herein comprise at least one of the CDR-Ls or variants thereof described in Table 18, wherein the multispecific molecules are capable of binding at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NOs: 212-213 and 443. In some embodiments, the multispecific molecules described herein comprise any one of the CDR-L1s or variants thereof described in Table 18, any one of the CDR-L2s or variants thereof described in Table 18, and any one of the CDR-L3s or variants thereof described in Table 18, wherein the multispecific molecules are capable of binding at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NOs: 212-213 and 443. In some embodiments, the multispecific molecules described herein comprise any combination of the CDR-L or variants thereof described in Table 18, wherein the multispecific molecules are capable of binding to at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NOs: 212-213 and 443. In some embodiments, the CDR-L variants comprise at least one, at least two, or at least three substitutions, deletions, additions, or combinations thereof relative to the corresponding parental CDR-L sequence described in Table 18. In some embodiments, the CDR-L or variants thereof comprise 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% of the same amino acid sequence as the corresponding parental CDR-L sequence described in Table 18.

[0162] In some embodiments, the engineered protein constructs described herein include an IL-12 binding moiety, wherein (a) the IL-12 binding moiety includes any one of the combinations of CDR-L1, CDR-L2 and CDR-L3 described in Table 18, and (b) the IL-12 binding moiety can bind at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% of the same amino acid sequence as any one of SEQ ID NO: 212-213 and 443. In some embodiments, the engineered protein constructs described herein include an IL-12 binding region and a TREM1 binding region, wherein (a) the IL-12 binding region includes any one of the combinations of CDR-L1, CDR-L2, and CDR-L3 described in Table 18, (b) the IL-12 binding region can bind at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NO: 212-213 and 443, and (c) the TREM1 binding region can bind TREM1, its functional fragments, its variants, or combinations thereof. In some embodiments, the multispecific molecule described herein comprises an IL-12 binding domain and a TREM1 binding domain, wherein (a) the IL-12 binding domain comprises any one of the combinations of CDR-L1, CDR-L2 and CDR-L3 described in Table 18, (b) the IL-12 binding domain can bind at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% of the same amino acid sequence as any one of SEQ ID NO:212-213 and 443, and (c) the TREM1 binding domain can bind TREM1, its functional fragments, its variants or combinations thereof.

[0163] Table 18. Combinations of CDR-Ls for binding to IL-12 family proteins

[0164] In some embodiments, the multispecific molecules described herein comprise combinations of CDRs, wherein the CDRs comprise 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 wherein the combination is any one of the combinations provided in Table 19.

[0165] Table 19. Exemplary CDR combinations for antibodies targeting IL-12 family proteins

[0166] In some embodiments, the multispecific molecule described herein comprises at least one VL sequence, wherein the multispecific molecule is capable of binding at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NOs: 212-213, 234, and 443-444. In some embodiments, the multispecific molecule described herein comprises 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% of the same amino acid sequence as any one of the VL sequences described in Table 20, wherein the multispecific molecule is capable of binding at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NOs: 212-213 and 443. In some embodiments, the engineered protein construct described herein includes an IL-12 binding moiety, wherein (a) the IL-12 binding moiety contains 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% of the same amino acid sequence as any one of the VL sequences described in Table 20, and (b) the IL-12 binding moiety can bind to at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NOs: 212-213 and 443. In some embodiments, the engineered protein constructs described herein include an IL-12 binding region and a TREM1 binding region, wherein (a) the IL-12 binding region contains 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% of the same amino acid sequence as any one of the VL sequences described in Table 20; (b) the IL-12 binding region may bind at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NO: 212-213 and 443; and (c) the TREM1 binding region may bind TREM1, its functional fragments, its variants, or combinations thereof.In some embodiments, the multispecific molecules described herein comprise an IL-12 binding domain and a TREM1 binding domain, wherein (a) the IL-12 binding domain comprises 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% of the same amino acid sequence as any one of the VL sequences described in Table 20, (b) the IL-12 binding domain can bind at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NOs: 212-213 and 443, and (c) the TREM1 binding domain can bind TREM1, its functional fragments, its variants, or combinations thereof.

[0167] Table 20. Exemplary VL sequences for binding to IL-12 family proteins

[0168] In some embodiments, the multispecific molecule described herein comprises at least one light chain (LC) region, wherein the multispecific molecule can bind at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NOs: 212-213, 234, and 443-444. In some embodiments, the multispecific molecule described herein comprises an IL-12 binding region, wherein the IL-12 binding region comprises an LC region. In some embodiments, the LC region of the IL-12 binding region comprises 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% of the same VL sequence as any one of the amino acid sequences described in Table 20.

[0169] In some embodiments, the LC region of the IL-12 binding region comprises a VL sequence and 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 RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 483), wherein the C-terminus of the VL sequence is linked to the N-terminus of the amino acid sequence.

[0170] In some embodiments, the multispecific molecule described herein comprises: (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 of the amino acid sequences described in Table 17; 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 of the amino acid sequences described in Table 20, wherein the multispecific molecule comprises a VH sequence and a VL sequence according to any of the combinations described in Table 21.

[0171] Table 21. Exemplary combinations of VH and VL sequences for binding to IL-12 family proteins.

[0172] IL-23 family As described herein, the IL-23 family includes interleukin-23α cytokine (IL-23α), its fragments, variants, multimeric forms, or combinations thereof. In some embodiments, IL-23 family proteins bind to the interleukin-23 receptor (IL23R). The amino acid sequences of IL-23α and IL23R are listed in Table 22.

[0173] Table 22. Amino acid sequences of IL-23 family proteins

[0174] In some embodiments, the multispecific molecule described herein binds to an amino acid sequence that is at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to any of the sequences listed in Table 22. In some embodiments, the multispecific molecule described herein binds to an amino acid sequence that is at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to any of SEQ ID NO: 234 or 452. In some embodiments, the multispecific molecule described herein may bind to: (a) an amino acid sequence that is at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to any of SEQ ID NO: 234 and 452; and (b) TREM1, its functional fragments, its variants, or combinations thereof.

[0175] In some embodiments, the multispecific molecules described herein comprise at least one of the CDR-Hs or variants thereof described in Table 23, wherein the multispecific molecules are capable of binding at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as SEQ ID NO: 234. In some embodiments, the multispecific molecules described herein comprise any one of the CDR-H1s or variants thereof described in Table 23, any one of the CDR-H2s or variants thereof described in Table 23, and any one of the CDR-H3s or variants thereof described in Table 23, wherein the multispecific molecules are capable of binding at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as SEQ ID NO: 234. In some embodiments, the multispecific antibody described herein comprises any combination of CDR-H or variants thereof described in Table 23, wherein the multispecific molecule is capable of binding to at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as SEQ ID NO: 234. In some embodiments, the CDR-H variant comprises at least one, at least two, or at least three substitutions, deletions, additions, or combinations thereof relative to the corresponding parental CDR-H sequence described in Table 23. In some embodiments, the CDR-H or variant thereof comprises 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% of the same amino acid sequence as the corresponding parental CDR-H sequence described in Table 23.

[0176] In some embodiments, the engineered protein constructs described herein include an IL-23 binding moiety, wherein (a) the IL-23 binding moiety includes any combination of CDR-H1, CDR-H2 and CDR-H3 described in Table 23, and (b) the IL-23 binding moiety can bind at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% of the same amino acid sequence as SEQ ID NO: 234. In some embodiments, the engineered protein constructs described herein include an IL-23 binding region and a TREM1 binding region, wherein (a) the IL-23 binding region includes any one of the combinations of CDR-H1, CDR-H2, and CDR-H3 described in Table 23, (b) the IL-23 binding region can bind at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as SEQ ID NO: 234, and (c) the TREM1 binding region can bind TREM1, its functional fragments, its variants, or combinations thereof. In some embodiments, the multispecific molecule described herein comprises an IL-23 binding domain and a TREM1 binding domain, wherein (a) the IL-23 binding domain comprises any one of the combinations of CDR-H1, CDR-H2, and CDR-H3 described in Table 23, (b) the IL-23 binding domain can bind at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as SEQ ID NO: 234, and (c) the TREM1 binding domain can bind TREM1, its functional fragments, its variants, or combinations thereof.

[0177] Table 23. Combinations of CDR-H for binding to IL-23 family proteins

[0178] In some embodiments, the multispecific molecule described herein comprises at least one VH sequence, wherein the multispecific molecule is capable of binding at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NO: 234 and 452. In some embodiments, the multispecific molecule described herein comprises 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% of the same amino acid sequence as any one of the VH sequences described in Table 24, wherein the multispecific molecule is capable of binding at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as SEQ ID NO: 234. In some embodiments, the engineered protein construct described herein includes an IL-23 binding moiety, wherein (a) the IL-23 binding moiety contains 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% of the same amino acid sequence as any one of the VH sequences described in Table 24, and (b) the IL-23 binding moiety can bind at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as SEQ ID NO: 234. In some embodiments, the engineered protein constructs described herein include an IL-23 binding region and a TREM1 binding region, wherein (a) the IL-23 binding region contains 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% of the same amino acid sequence as any one of the VH sequences described in Table 24, (b) the IL-23 binding region can bind at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as SEQ ID NO:234, and (c) the TREM1 binding region can bind TREM1, its functional fragments, its variants, or combinations thereof.In some embodiments, the multispecific molecule described herein comprises an IL-23 binding domain and a TREM1 binding domain, wherein (a) the IL-23 binding domain comprises 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% of the amino acid sequence identical to any one of the VH sequences described in Table 24, (b) the IL-23 binding domain may bind at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the amino acid sequence identical to SEQ ID NO: 234, and (c) the TREM1 binding domain may bind TREM1, its functional fragments, its variants, or combinations thereof.

[0179] Table 24. Exemplary VH sequences for binding to IL-23 family proteins

[0180] In some embodiments, the multispecific molecule described herein comprises at least one heavy chain (HC) region, wherein the multispecific molecule can bind at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NO: 234 and 452. In some embodiments, the multispecific molecule described herein comprises an IL-23 binding region, wherein the IL-23 binding region comprises an HC region. In some embodiments, the HC region of the IL-23 binding region comprises 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% of the same VH sequence as any one of the amino acid sequences described in Table 24.

[0181] In some implementations, the HC region of the IL-23 binding region contains the VH sequence and the sequence ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 481) 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% of the same amino acid sequence, wherein the C-terminus of the VH sequence is connected to the N-terminus of the amino acid sequence.

[0182] In some implementations, the HC region of the IL-23 binding region contains the VH sequence and the sequence ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 482) 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% of the same amino acid sequence, wherein the C-terminus of the VH sequence is connected to the N-terminus of the amino acid sequence.

[0183] In some embodiments, the multispecific molecules described herein comprise at least one of the CDR-Ls or variants thereof described in Table 25, wherein the multispecific molecules are capable of binding at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as SEQ ID NO: 234. In some embodiments, the multispecific molecules described herein comprise any one of the CDR-L1s or variants thereof described in Table 25, any one of the CDR-L2s or variants thereof described in Table 25, and any one of the CDR-L3s or variants thereof described in Table 25, wherein the multispecific molecules are capable of binding at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as SEQ ID NO: 234. In some embodiments, the multispecific molecules described herein comprise any combination of the CDR-L or variants thereof described in Table 25, wherein the multispecific molecules are capable of binding to at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as SEQ ID NO: 234. In some embodiments, the CDR-L variants comprise at least one, at least two, or at least three substitutions, deletions, additions, or combinations thereof relative to the corresponding parental CDR-L sequence described in Table 25. In some embodiments, the CDR-L or variants thereof comprise 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% of the same amino acid sequence as the corresponding parental CDR-L sequence described in Table 25.

[0184] In some embodiments, the engineered protein constructs described herein include an IL-23 binding moiety, wherein (a) the IL-23 binding moiety includes any combination of CDR-L1, CDR-L2 and CDR-L3 described in Table 25, and (b) the IL-23 binding moiety can bind at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% of the same amino acid sequence as SEQ ID NO: 234. In some embodiments, the engineered protein constructs described herein include an IL-23 binding region and a TREM1 binding region, wherein (a) the IL-23 binding region includes any one of the combinations of CDR-L1, CDR-L2, and CDR-L3 described in Table 25, (b) the IL-23 binding region can bind at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as SEQ ID NO: 234, and (c) the TREM1 binding region can bind TREM1, its functional fragments, its variants, or combinations thereof. In some embodiments, the multispecific molecule described herein comprises an IL-23 binding domain and a TREM1 binding domain, wherein (a) the IL-23 binding domain comprises any one of the combinations of CDR-L1, CDR-L2, and CDR-L3 described in Table 25, (b) the IL-23 binding domain can bind at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as SEQ ID NO: 234, and (c) the TREM1 binding domain can bind TREM1, its functional fragments, its variants, or combinations thereof.

[0185] Table 25. Combinations of CDR-L for binding to IL-23 family proteins

[0186] In some embodiments, the multispecific molecules described herein comprise combinations of CDRs, wherein the CDRs comprise 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 wherein the combination is any one of the combinations provided in Table 26.

[0187] Table 26. Exemplary CDR combinations for antibodies targeting IL-23 family proteins

[0188] In some embodiments, the multispecific molecule described herein comprises at least one VL sequence, wherein the multispecific molecule is capable of binding at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NO: 234 and 452. In some embodiments, the multispecific molecule described herein comprises 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% of the same amino acid sequence as any one of the VL sequences described in Table 27, wherein the multispecific molecule is capable of binding at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as SEQ ID NO: 234. In some embodiments, the engineered protein construct described herein includes an IL-23 binding moiety, wherein (a) the IL-23 binding moiety contains 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% of the same amino acid sequence as any one of the VL sequences described in Table 27, and (b) the IL-23 binding moiety can bind at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as SEQ ID NO: 234. In some embodiments, the engineered protein constructs described herein include an IL-23 binding region and a TREM1 binding region, wherein (a) the IL-23 binding region contains 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% of the amino acid sequence identical to any one of the VL sequences described in Table 27, (b) the IL-23 binding region can bind at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the amino acid sequence identical to SEQ ID NO:234, and (c) the TREM1 binding region can bind TREM1, its functional fragments, its variants, or combinations thereof.In some embodiments, the multispecific molecule described herein comprises an IL-23 binding domain and a TREM1 binding domain, wherein (a) the IL-23 binding domain comprises 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% of the amino acid sequence identical to any one of the VL sequences described in Table 27, (b) the IL-23 binding domain may bind at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the amino acid sequence identical to SEQ ID NO: 234, and (c) the TREM1 binding domain may bind TREM1, its functional fragments, its variants, or combinations thereof.

[0189] Table 27. Exemplary VL sequences for binding to IL-23 family proteins

[0190] In some embodiments, the multispecific molecule described herein comprises at least one light chain (LC) region, wherein the multispecific molecule can bind at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NO: 234 or 452. In some embodiments, the multispecific molecule described herein comprises an IL-23 binding region, wherein the IL-23 binding region comprises an LC region. In some embodiments, the LC region of the IL-23 binding region comprises 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% of the same VL sequence as any one of the amino acid sequences described in Table 27.

[0191] In some embodiments, the LC region of the IL-23 binding region comprises a VL sequence and 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 RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 483), wherein the C-terminus of the VL sequence is linked to the N-terminus of the amino acid sequence.

[0192] In some embodiments, the multispecific molecule described herein comprises: (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 of the amino acid sequences described in Table 24; 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 of the amino acid sequences described in Table 27, wherein the multispecific molecule comprises a VH sequence and a VL sequence according to any of the combinations described in Table 28.

[0193] Table 28. Exemplary combinations of VH and VL sequences for binding to IL-23 family proteins.

[0194] TREM1 Triggering Receptor Expressed on Myeloid Cells 1 (TREM1, also known as CD354, HGNC: 17760, Entrez Gene: 54210, UniProtKB: Q9NP99), expressed on myeloid cells, belongs to the Ig receptor superfamily and is highly expressed on myeloid cell subsets, including neutrophils, monocytes, and macrophages. TREM1 amplifies cell surface receptors involved in inflammatory responses and innate and adaptive immune functions. TREM1 does not contain its own signaling motif but is activated via the adaptor DAP 12 (DNAX activator protein 12). This can lead to an amplification of the inflammatory response (Bouchon et al. (2000) J. Immunol. 164 (10):4991-4995). Crosslinking of TREM1 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 acute inflammatory responses during septic shock and infection (Cohen, (2001) Lancet. 358:776-778). Furthermore, TREM1 is associated with other inflammatory 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. NatRev Immunol (2023). https: / / doi.org / 10.1038 / s41577-023-00837-1). In some embodiments, PGLYRP1 (peptidoglycan recognition protein 1) is molecularly targeted by the molecules described herein to bind TREM1. Five activated forms of the TREM receptor exist, including TREM 1, 2, 3, 4, and 5, with the soluble form TREM1 (sTREM1) released during infection. TREM1 consists of a single immunoglobulin V (Ig-V)-like domain 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 fragments thereof. In some cases, the bispecific molecules described herein bind to specific domains of TREM1 or sTREM1, such as the Ig-V domain or the stem region.

[0195] In some embodiments, the multispecific molecules described herein are capable of binding to the TREM1 protein, including antibodies that incapacitate unstimulated myeloid cells. In some embodiments, the multispecific (e.g., bispecific, trispecific) molecules described herein bind to the mammalian TREM1 sequence. In some cases, TREM1 is a mouse homolog.

[0196] The amino acid sequence of the human TREM1 protein is listed in Table 29.

[0197] Table 29. Amino acid sequence of human TREM1 protein

[0198] In some embodiments, the multispecific molecules described herein bind to an amino acid sequence that is at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to any of the sequences listed in Table 29. In some embodiments, the multispecific molecules described herein bind to an amino acid sequence that is at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to any of SEQ ID NO: 398 and 456-458. In some embodiments, the multispecific molecular binding described herein includes: (a) an amino acid sequence that is at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to any one of SEQ ID NO: 398 and 456-458; and (b) a protein, a variant thereof, a functional fragment thereof, or a combination thereof selected from any one of the IL-1 family, IL-6 family, IL-12 family, and IL-23 family.

[0199] In some embodiments, the multispecific molecules described herein comprise at least one of the CDR-Hs or variants thereof described in Table 30, wherein the multispecific molecules are capable of binding at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NO: 398 and 456-458. In some embodiments, the multispecific molecules described herein comprise any one of the CDR-H1s or variants thereof described in Table 30, any one of the CDR-H2s or variants thereof described in Table 30, and any one of the CDR-H3s or variants thereof described in Table 30, wherein the multispecific molecules are capable of binding at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NO: 398 and 456-458. In some embodiments, the multispecific molecules described herein comprise any combination of CDR-H or variants thereof described in Table 30.1, wherein the multispecific molecules are capable of binding to at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NO: 398 and 456-458. In some embodiments, the CDR-H variants comprise at least one, at least two, or at least three substitutions, deletions, additions, or combinations thereof relative to the corresponding parental CDR-H sequence described in Table 30. In some embodiments, the CDR-H or variants thereof comprise 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% of the same amino acid sequence as the corresponding parental CDR-H sequence described in Table 30.

[0200] Table 30. CDR-H for use with TREM1

[0201] In some embodiments, the engineered protein constructs described herein include a TREM1 binding moiety, wherein (a) the TREM1 binding moiety includes any combination of CDR-H1, CDR-H2 and CDR-H3 described in Table 30.1, and (b) the TREM1 binding moiety can bind at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% of the same amino acid sequence as any one of SEQ ID NO: 398 and 456-458. In some embodiments, the engineered protein constructs described herein include a TREM1 binding region and an interleukin binding region, wherein (a) the TREM1 binding region includes any one of the combinations of CDR-H1, CDR-H2, and CDR-H3 described in Table 30.1, (b) the TREM1 binding region can bind an amino acid sequence that is at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to any one of SEQ ID NO: 398 and 456-458, and (c) the interleukin binding region can bind a protein, its variants, its functional fragments, or a combination thereof selected from any one of the IL-1 family, IL-6 family, IL-12 family, and IL-23 family. In some embodiments, the multispecific molecule described herein comprises a TREM1 binding domain and an interleukin binding domain, wherein (a) the TREM1 binding domain comprises any one of the combinations of CDR-H1, CDR-H2 and CDR-H3 described in Table 30.1, (b) the TREM1 binding domain can bind an amino acid sequence that is at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identical to any one of SEQ ID NO: 398 and 456-458, and (c) the interleukin binding domain can bind a protein, a variant thereof, a functional fragment thereof or a combination thereof selected from any one of the IL-1 family, IL-6 family, IL-12 family and IL-23 family.

[0202] Table 30.1. Combinations of CDR-H for use with TREM1

[0203] In some embodiments, the multispecific molecule described herein comprises at least one VH sequence, wherein the multispecific molecule is capable of binding at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NO: 398 and 456-458. In some embodiments, the multispecific molecule described herein comprises 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% of the same amino acid sequence as any one of the VH sequences described in Table 31, wherein the multispecific molecule is capable of binding at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as any one of SEQ ID NO: 398 and 456-458. In some embodiments, the engineered protein constructs described herein include a TREM1 binding moiety, wherein (a) the TREM1 binding moiety contains 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% of the same amino acid sequence as any one of the VH sequences described in Table 31, and (b) the TREM1 binding moiety can bind at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NO: 398 and 456-458. In some embodiments, the engineered protein constructs described herein include a TREM1 binding region and an interleukin binding region, wherein (a) the TREM1 binding region contains 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% of the same amino acid sequence as any one of the VH sequences described in Table 31, (b) the TREM1 binding region can bind at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NO: 398 and 456-458, and (c) the interleukin binding region can bind proteins, variants thereof, functional fragments thereof, or combinations thereof selected from any one of the IL-1 family, IL-6 family, IL-12 family, and IL-23 family.In some embodiments, the multispecific molecule described herein comprises a TREM1 binding domain and an interleukin binding domain, wherein (a) the TREM1 binding domain comprises 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% of the same amino acid sequence as any one of the VH sequences described in Table 31, (b) the TREM1 binding domain can bind at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NO: 398 and 456-458, and (c) the interleukin binding domain can bind proteins, variants thereof, functional fragments thereof, or combinations thereof selected from any one of the IL-1 family, IL-6 family, IL-12 family, and IL-23 family.

[0204] Table 31. Exemplary VH sequences for binding with TREM1

[0205] In some embodiments, the multispecific molecule described herein comprises at least one heavy chain (HC) region, wherein the multispecific molecule can bind at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NO: 398 and 456-458. In some embodiments, the multispecific molecule described herein comprises a TREM1 binding region, wherein the TREM1 binding region comprises an HC region. In some embodiments, the HC region of the TREM1 binding region comprises 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% of the same VH sequence as any one of the amino acid sequences described in Table 31.

[0206] In some implementations, the HC region of the TREM1 binding region contains the VH sequence and the sequence ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 481) 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% of the same amino acid sequence, wherein the C-terminus of the VH sequence is connected to the N-terminus of the amino acid sequence.

[0207] In some implementations, the HC region of the TREM1 binding region contains the VH sequence and the sequence ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 482) 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% of the same amino acid sequence, wherein the C-terminus of the VH sequence is connected to the N-terminus of the amino acid sequence.

[0208] In some embodiments, the multispecific molecules described herein comprise at least one of the CDR-Ls or variants thereof described in Table 32, wherein the multispecific molecules are capable of binding at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NO: 398 and 456-458. In some embodiments, the multispecific molecules described herein comprise any one of the CDR-L1s or variants thereof described in Table 32, any one of the CDR-L2s or variants thereof described in Table 32, and any one of the CDR-L3s or variants thereof described in Table 32, wherein the multispecific molecules are capable of binding at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NO: 398 and 456-458. In some embodiments, the multispecific molecules described herein comprise any combination of the CDR-L or variants thereof described in Table 32.1, wherein the multispecific molecules are capable of binding to at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NO: 398 and 456-458. In some embodiments, the CDR-L variants comprise at least one, at least two, or at least three substitutions, deletions, additions, or combinations thereof relative to the corresponding parental CDR-L sequence described in Table 32. In some embodiments, the CDR-L or variants thereof comprise 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% of the same amino acid sequence as the corresponding parental CDR-L sequence described in Table 32.

[0209] Table 32. CDR-L for use with TREM1

[0210] In some embodiments, the engineered protein constructs described herein include a TREM1 binding moiety, wherein (a) the TREM1 binding moiety includes any combination of CDR-L1, CDR-L2 and CDR-L3 described in Table 32.1, and (b) the TREM1 binding moiety can bind at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% of the same amino acid sequence as any one of SEQ ID NO: 398 and 456-458. In some embodiments, the engineered protein constructs described herein include a TREM1 binding region and an interleukin binding region, wherein (a) the TREM1 binding region includes any one of the combinations of CDR-L1, CDR-L2, and CDR-L3 described in Table 32.1, (b) the TREM1 binding region can bind an amino acid sequence that is at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to any one of SEQ ID NO: 398 and 456-458, and (c) the interleukin binding region can bind a protein, a variant thereof, a functional fragment thereof, or a combination thereof selected from any one of the IL-1 family, IL-6 family, IL-12 family, and IL-23 family. In some embodiments, the multispecific molecule described herein comprises a TREM1 binding domain and an interleukin binding domain, wherein (a) the TREM1 binding domain comprises any one of the combinations of CDR-L1, CDR-L2 and CDR-L3 described in Table 32.1, (b) the TREM1 binding domain can bind an amino acid sequence that is at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identical to any one of SEQ ID NO: 398 and 456-458, and (c) the interleukin binding domain can bind a protein, a variant thereof, a functional fragment thereof or a combination thereof selected from any one of the IL-1 family, IL-6 family, IL-12 family and IL-23 family.

[0211] Table 32.1. Combinations of CDR-L for use with TREM1

[0212] In some embodiments, the multispecific molecules described herein comprise combinations of CDRs, wherein the CDRs comprise 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 wherein the combination is any one of the combinations provided in Table 33.

[0213] Table 33. Exemplary CDR combinations for antibodies targeting TREM1

[0214] In some embodiments, the multispecific molecule described herein comprises at least one VL sequence, wherein the multispecific molecule is capable of binding to at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the amino acid sequence identical to any one of the amino acid sequences in Table 29. In some embodiments, the multispecific molecule described herein comprises 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% of the amino acid sequence identical to any one of the VL sequences described in Table 34, wherein the multispecific molecule is capable of binding to at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the amino acid sequence identical to any one of SEQ ID NO: 398 and 456-458. In some embodiments, the engineered protein constructs described herein include a TREM1 binding moiety, wherein (a) the TREM1 binding moiety contains 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% of the same amino acid sequence as any one of the VL sequences described in Table 34, and (b) the TREM1 binding moiety can bind to at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NO: 398 and 456-458. In some embodiments, the engineered protein constructs described herein include a TREM1 binding region and an interleukin binding region, wherein (a) the TREM1 binding region contains 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% of the same amino acid sequence as any one of the VL sequences described in Table 34, (b) the TREM1 binding region can bind at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NO: 398 and 456-458, and (c) the interleukin binding region can bind a protein, its variants, its functional fragments, or a combination thereof selected from any one of the IL-1 family, IL-6 family, IL-12 family, and IL-23 family.In some embodiments, the multispecific molecule described herein comprises a TREM1 binding domain and an interleukin binding domain, wherein (a) the TREM1 binding domain comprises 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% of the same amino acid sequence as any one of the VL sequences described in Table 34, (b) the TREM1 binding domain can bind at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NO: 398 and 456-458, and (c) the interleukin binding domain can bind proteins, variants thereof, functional fragments thereof, or combinations thereof selected from any one of the IL-1 family, IL-6 family, IL-12 family, and IL-23 family.

[0215] Table 34. Exemplary VL sequences for binding with TREM1

[0216] In some embodiments, the multispecific molecule described herein comprises at least one light chain (LC) region, wherein the multispecific molecule can bind at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NO: 398 and 456-458. In some embodiments, the multispecific molecule described herein comprises a TREM1 binding region, wherein the TREM1 binding region comprises an LC region. In some embodiments, the LC region of the TREM1 binding region comprises 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% of the same VL sequence as any one of the amino acid sequences described in Table 34.

[0217] In some embodiments, the LC region of the TREM1 binding region comprises a VL sequence and 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 RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 483), wherein the C-terminus of the VL sequence is linked to the N-terminus of the amino acid sequence.

[0218] In some embodiments, the multispecific molecule described herein comprises: (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 of the amino acid sequences described in Table 31; 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 of the amino acid sequences described in Table 34, wherein the multispecific molecule comprises a VH sequence and a VL sequence in combination as described in Table 35.

[0219] Table 35. Exemplary combinations of VH and VL sequences for binding with TREM1

[0220] In some embodiments, the multispecific molecule described herein comprises at least one heavy chain (HC) region, wherein the multispecific molecule is capable of binding at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NO: 398 and 456-458. In some embodiments, the multispecific molecule described herein comprises 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% of the same amino acid sequence as any one of the HC regions described in Table 36, wherein the multispecific molecule is capable of binding at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NO: 398 and 456-458. In some embodiments, the engineered protein constructs described herein include a TREM1 binding moiety, wherein (a) the TREM1 binding moiety contains 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% of the amino acid sequence of any one of the HC regions described in Table 36, and (b) the TREM1 binding moiety may bind to at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the amino acid sequence of any one of SEQ ID NO: 398 and 456-458. In some embodiments, the engineered protein constructs described herein include a TREM1 binding region and an interleukin binding region, wherein (a) the TREM1 binding region contains 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% of the amino acid sequence of any one of the HC regions described in Table 36, (b) the TREM1 binding region can bind at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the amino acid sequence of any one of SEQ ID NO: 398 and 456-458, and (c) the interleukin binding region can bind proteins, variants thereof, functional fragments thereof, or combinations thereof selected from any one of the IL-1 family, IL-6 family, IL-12 family, and IL-23 family.In some embodiments, the multispecific molecule described herein comprises a TREM1 binding domain and an interleukin binding domain, wherein (a) the TREM1 binding domain comprises 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% of the amino acid sequence of any one of the HC regions described in Table 36, (b) the TREM1 binding domain can bind at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the amino acid sequence of any one of SEQ ID NO: 398 and 456-458, and (c) the interleukin binding domain can bind proteins, variants thereof, functional fragments thereof, or combinations thereof selected from any one of the IL-1 family, IL-6 family, IL-12 family, and IL-23 family.

[0221] Table 36. Exemplary HC regions of TREM1 combined with structural domains

[0222] In some embodiments, the multispecific molecule described herein comprises at least one light chain (LC) sequence, wherein the multispecific molecule is capable of binding at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NO: 398 and 456-458. In some embodiments, the multispecific molecule described herein comprises 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% of the same amino acid sequence as any one of the LC regions described in Table 37, wherein the multispecific molecule is capable of binding at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the same amino acid sequence as any one of SEQ ID NO: 398 and 456-458. In some embodiments, the engineered protein construct described herein includes a TREM1 binding moiety, wherein (a) the TREM1 binding moiety contains 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% of the amino acid sequence of any one of the LC regions described in Table 37, and (b) the TREM1 binding moiety can bind to at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the amino acid sequence of any one of SEQ ID NO: 398 and 456-458. In some embodiments, the engineered protein constructs described herein include a TREM1 binding region and an interleukin binding region, wherein (a) the TREM1 binding region contains 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% of the amino acid sequence of any one of the LC regions described in Table 37; (b) the TREM1 binding region can bind at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the amino acid sequence of any one of SEQ ID NO: 398 and 456-458; and (c) the interleukin binding region can bind a protein, its variants, its functional fragments, or a combination thereof selected from any one of the IL-1 family, IL-6 family, IL-12 family, and IL-23 family.In some embodiments, the multispecific molecule described herein comprises a TREM1 binding domain and an interleukin binding domain, wherein (a) the TREM1 binding domain comprises 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% of the amino acid sequence of any one of the LC regions described in Table 37, (b) the TREM1 binding domain can bind at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the amino acid sequence of any one of SEQ ID NO: 398 and 456-458, and (c) the interleukin binding domain can bind proteins, variants thereof, functional fragments thereof, or combinations thereof selected from any one of the IL-1 family, IL-6 family, IL-12 family, and IL-23 family.

[0223] Table 37. Exemplary LC regions of TREM1 combined with structural domains

[0224] In some embodiments, the multispecific antibody described herein comprises: (a) an HC region 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 of the amino acid sequences described in Table 36; and (b) an LC region 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 of the amino acid sequences described in Table 37, wherein the HC region and the LC region are selected according to the combination described in Table 38.

[0225] Table 38. Exemplary combinations of HC and LC regions for use with TREM1

[0226] Nucleotide constructs of BsAb This document provides nucleotide sequences encoding the TREM1 binding domain, its variants, or functional fragments thereof. In some embodiments, the TREM1 binding domain comprises an HC region, an LC region, or a combination thereof. In some embodiments, the nucleotide sequence encodes the HC region of the TREM1 binding domain, wherein the HC region comprises any one of the combinations of CDR-H or its variants described in Table 30.1, wherein the variants comprise 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% of the amino acid sequence identical to the corresponding parental CDR-H sequence described in Table 30.1. In some embodiments, the nucleotide sequence encodes the HC region of the TREM1 binding domain, wherein the HC region comprises a VH sequence, its variants, or functional fragments thereof, wherein the VH sequence comprises any one of the sequences described in Table 31, and wherein the variants comprise 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% of the amino acid sequence identical to the corresponding parental VH sequence described in Table 31. In some embodiments, the nucleotide sequence encodes the HC region of the TREM1 binding domain, wherein the HC region comprises the amino acid sequence described in Table 36, and wherein the variant comprises 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% of the amino acid sequence identical to the corresponding parental HC sequence described in Table 36. In some embodiments, the nucleotide sequence encodes the LC region of the TREM1 binding domain, wherein the LC region comprises any combination of the CDR-L or variants described in Table 32.1, wherein the variant comprises 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% of the amino acid sequence identical to the corresponding parental CDR-L sequence described in Table 32.1. In some embodiments, the nucleotide sequence encodes the LC region of the TREM1 binding domain, wherein the LC region comprises a VL sequence, a variant thereof, or a functional fragment thereof, wherein the VL sequence comprises any of the sequences described in Table 34, and wherein the variant comprises 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% of the amino acid sequence identical to the corresponding parental VL sequence described in Table 34. In some embodiments, the nucleotide sequence encodes the LC region of the TREM1 binding domain, wherein the LC region comprises the amino acid sequence described in Table 37, wherein the variant comprises 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% of the amino acid sequence identical to the corresponding parental HC sequence described in Table 37.

[0227] Table 39 provides exemplary nucleotide sequences of some proteins or portions thereof described herein. In some embodiments, the nucleotide sequence encoding BsAb or portions thereof comprises 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 described in Table 39.

[0228] Table 39. Exemplary nucleotide sequences encoding BsAb or a portion thereof

[0229] BsAb This article discloses a bispecific antibody (BsAb) that binds to TREM1 and interleukins (e.g., proteins, variants or functional fragments thereof selected from any one of the IL-1, IL-6, IL-12 and IL-23 families). Figure 1 A bispecific antibody binding TREM1 and interleukin is described. In some embodiments, the BsAb contains a heavy chain region. In some embodiments, the BsAb contains a light chain region. In some embodiments, the light chain region contains a κ light chain constant region. In some embodiments, the BsAb contains a BsAb light chain variable region. In some embodiments, the BsAb contains a BsAb heavy chain variable region. In some embodiments, the BsAb contains both 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 contains a common light chain (L chain). In some embodiments, the L chain acts on a specific antigen. The use of the common light chain can preferentially promote heterodimerization in the Fc region. In some embodiments, the L chain contains a node mutation. In some embodiments, the L chain contains a pore mutation. In some embodiments, heterodimerization is preferentially formed when binding to a BsAb containing a common L chain. In some implementations, the formation of heterodimer pairs is achieved using glutathione disulfide bond exchange for assembly.

[0230] This document discloses engineered protein molecules. In some embodiments, the engineered protein molecules described herein comprise the BsAbs described herein. In some embodiments, the BsAbs described herein are engineered BsAbs comprising one or more amino acid modifications that can result in pH-dependent target-binding activity. In some embodiments, the engineered BsAbs described herein can exhibit pH-dependent target-binding activity against target peptides selected from TREM1, interleukins (e.g., IL-1 family, IL-6 family, IL-12 family, IL-23 family proteins), their variants, and functional fragments thereof. In some embodiments, the engineered BsAbs described herein can readily bind to the target peptide at neutral pH and dissociate from the target peptide at acidic pH. Therefore, upon administration to a subject, the engineered BsAb can bind to the target peptide in plasma due to its neutral pH, while remaining dissociated from the target peptide in endosomes with an acidic pH. The dissociation of the engineered BsAb from the target peptide in endosomes can facilitate the recycling of the engineered BsAb into plasma via FcRn, while the target peptide can be transported to lysosomes and degraded. Such features of engineered BsAbs can allow for the removal of target peptides from plasma. Therefore, in some embodiments, engineered BsAbs may contain target peptide removal activity against target peptides selected from TREM1, interleukins (e.g., IL-1 family, IL-6 family, IL-12 family, IL-23 family proteins), their variants, and functional fragments thereof.

[0231] In some embodiments, the BsAb described herein is an engineered BsAb comprising one or more amino acid modifications that result in increased FcRn binding at neutral pH. In such embodiments, the engineered BsAb may have increased ability to repeatedly bind to FcRn and remove target peptides from plasma. Optionally, in some embodiments, the BsAb described herein is an engineered BsAb comprising one or more amino acid modifications that result in increased FcRn binding at acidic pH. In such embodiments, the engineered BsAb may have increased recycling efficiency from endosomal to plasma, resulting in improved plasma retention of the engineered BsAb. Therefore, in some embodiments, the constant domains of the engineered BsAb as described herein may be further modified to increase FcRn binding activity at neutral and / or acidic pH.

[0232] In some embodiments, the BsAb described herein is an engineered BsAb comprising one or more amino acid modifications that can cause a change in the isoelectric point of the BsAb. In some embodiments, one or more amino acid modifications can cause a change in the isoelectric point of the VH sequence of the engineered BsAb. In some embodiments, one or more amino acid modifications can cause a change in the isoelectric point of the VL sequence of the engineered BsAb. In some embodiments, one or more amino acid modifications can increase the isoelectric point of the engineered BsAb. In some embodiments, the increased isoelectric point can lead to an increased rate of elimination of the target peptide from plasma.

[0233] In some embodiments, the BsAb described herein is an engineered BsAb comprising one or more amino acid modifications that can result in pH-dependent target binding activity and / or increased FcRn binding activity. In some embodiments, the one or more amino acid modifications include 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 amino acid modifications. In some embodiments, the one or more amino acid modifications are in at least one of the Fab region, scFv region, and Fc region. In some embodiments, the one or more amino acid modifications are in the VL sequence of the BsAb, the VH sequence of the BsAb, or a combination thereof. In some embodiments, the amino acid modification is the deletion, substitution, or addition of an amino acid.

[0234] In some embodiments, BsAb may be a monospecific antibody, including but not limited to antibodies whose two arms target different epitopes of the same antigen. In some embodiments, BsAb may be a bispecific antibody. In some embodiments, BsAb may be a trispecific antibody. In some embodiments, BsAb may be a multispecific antibody.

[0235] In some embodiments, the BsAb described herein induces TREM1 degradation, TREM1 cleavage, TREM1 internalization, TREM1 shedding, TREM1 expression downregulation, or a combination thereof. In some embodiments, the BsAb described herein inhibits the interaction (e.g., binding) between TREM1 and one or more TREM1 ligands. In some embodiments, the BsAb described herein is transiently activated and then induces one or more of the following: TREM1 degradation, TREM1 cleavage, TREM1 internalization, TREM1 shedding, TREM1 expression downregulation, and TREM1 expression reduction.

[0236] In some embodiments, the BsAbs described herein bind to pro-inflammatory receptors and pro-inflammatory cytokines. Pro-inflammatory receptors enhance immune responses. For example, TREM1 is a pro-inflammatory receptor that, upon activation, induces the production of pro-inflammatory cytokines such as IL-1, IL-2, IL-6, IL-8, IL-12, IL-23, and TNF-α; chemokines such as MIP-1α, membrane cofactors 1 and 2, and GM-CSF; and co-stimulatory molecules such as CD1a, CD86, and MHC class II. Therefore, TREM1 is associated with the occurrence of immune-related inflammatory diseases. Decreased TREM1 activity can lead to a decrease / attenuation of pro-inflammatory cytokine production. Therefore, in some embodiments, treatment with the BsAbs described herein advantageously reduces inflammatory effects through two independent mechanisms: (1) by directly inhibiting inflammatory activity through binding to pro-inflammatory cytokines, and (2) by indirectly inhibiting inflammatory activity by reducing the production of pro-inflammatory cytokines through binding to TREM1.

[0237] In some embodiments, administration of the BsAb containing the interleukin-binding region described herein results in decreased TREM-1 expression in subjects with autoimmune conditions. Therefore, in some embodiments, administration of the BsAb described herein to subjects with autoimmune conditions can advantageously reduce TREM1 activity through two independent mechanisms: (1) by directly inhibiting TREM1 activity through binding to TREM1, and (2) by indirectly inhibiting TREM1 activity by reducing TREM1 expression through decreasing interleukin activity.

[0238] In some implementations, administration of BsAb containing the TREM-1 binding region described herein leads to increased expression of TREM-1-related genes. In some implementations, TREM-1-related genes include nicotinamide phosphoribosyltransferase (NAMPT), dehydrogenase / reductase 9 (DHRS9), cyclin-dependent kinase inhibitor 1A (CDKN1A), CD52 molecule (CD52), myotubulin-associated protein 11 (MTMR11), EH domain-containing 1 (EHD1), solute carrier family 27 member 3 (SLC27A3), interleukin 24 (IL24), Pim-2 proto-oncogene serine / threonine kinase (PIM2), chitosanase 3-like 1 (CHI3L1), polypeptide N-acetylgalactosyltransferase 6 (GALNT6), acyl-CoA thioesterase 7 (ACOT7), protein containing cytokine-inducible SH2 (CISH), sequence-similar family 129 member A (FAM129A), polo-like kinase 3 (PLK3), and a major facilitater superfamily 12. (MFSD12), containing StAR-associated lipid transfer domain 4 (STARD4), member A of family 12 of c-type lectin domain (CLEC12A), CD55 molecule (Cromer blood group) (CD55), interferon λ receptor 1 (IFNLR1) or a combination thereof.

[0239] High TREM1 activity in peripheral myeloid and / or microglia can be a cause of age-related inflammation and cognitive decline. TREM1 inhibits the expression of genes associated with key enzymes in the pentose phosphate pathway, leading to a deficiency in ribose-5-phosphate. Ribose-5-phosphate is an essential intermediate in the synthesis of purines and pyrimidines. This deficiency can be corrected by reducing TREM1 activity. Therefore, in some embodiments, administration of a BsAb containing the TREM-1 binding region described herein restores the pentose phosphate pathway (PPP). In some embodiments, administration of a BsAb containing the TREM-1 binding region described herein restores the expression efficiency of the glycolytic transcripts hexokinase and pyruvate kinase. In some embodiments, administration of a 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.

[0240] In some embodiments, the BsAb described herein comprises at least as much anti-inflammatory activity as a combination of a monospecific antibody binding to TREM1 and a monospecific antibody binding to interleukins (e.g., proteins, variants thereof, or functional fragments thereof selected from the IL-1, IL-6, IL-12, or IL-23 families). In some embodiments, the BsAb advantageously reduces the need for administration of two separate antibodies. In some embodiments, subjects treated with the BsAb advantageously tolerate higher doses compared to the combination of monospecific antibodies.

[0241] In some embodiments, BsAb binds TREM1 and interleukins (e.g., proteins, variants thereof, or functional fragments thereof selected from the IL-1, IL-6, IL-12, or IL-23 families), wherein the BsAb contains an anti-inflammatory activity of 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, relative to the combined anti-inflammatory activity of the monospecific antibody binding TREM1 and the monospecific antibody binding interleukins.

[0242] Furthermore, the BsAb described herein comprises two therapeutic domains (a TREM1-binding domain and an interleukin-binding domain) for each Fc region. In contrast, monospecific antibodies comprise only one therapeutic domain (either a TREM1-binding domain or an interleukin-binding domain) for each Fc region. Therefore, administration of BsAb advantageously reduces the amount of Fc region applied. In some embodiments, BsAb advantageously exhibits fewer side effects compared to administration of two separate antibodies. In some embodiments, the BsAb described herein advantageously comprises a combination of a TREM1-binding monospecific antibody and an interleukin-binding monospecific antibody (e.g., IL-1 family, IL-6 family, IL-12 family, IL-23 family) exhibiting higher anti-inflammatory activity.

[0243] Treatment with interleukin-binding antibodies to reduce interleukin-mediated inflammatory responses in subjects can cause upper respiratory tract infections, headache, nausea, vomiting, rectal bleeding, sore throat, candidiasis, hypersensitivity reactions, hypertension, diarrhea, back pain, cough, malignancy, and major adverse cardiovascular events. For example, suppressing interleukin activity in subjects to treat inflammatory conditions can cause candidiasis. In some embodiments, the incidence (or occurrence) of one or more side effects associated with reduced interleukin activity is lower in subjects treated with the BsAb described herein to treat inflammatory conditions (or symptoms) compared to subjects treated with monospecific interleukin-binding antibodies that reduce interleukin activity. For example, in some embodiments, treatment with the BsAb described herein reduces the incidence of candidiasis in subjects with inflammatory conditions compared to subjects treated with monospecific antibodies that bind interleukins and / or reduce interleukin activity.

[0244] In some embodiments, the BsAbs described herein are engineered to possess pH-dependent target-binding activity. In some embodiments, the BsAbs comprise such engineered BsAbs. In some embodiments, the engineered BsAbs possess pH-dependent target-binding activity against at least one target, wherein the at least one target is selected from TREM1 and interleukins. In some embodiments, the engineered BsAbs readily bind to at least one target at neutral pH and dissociate from at least one target at acidic pH. Therefore, the engineered BsAbs bind to at least one target in plasma with a neutral pH and dissociate from at least one target in endosomes with an acidic pH. Dissociation of the engineered BsAb from at least one target in endosomes allows the engineered BsAb to be recycled back into plasma via FcRn. However, at least one target is transported to lysosomes and degraded. Such characteristics of the engineered BsAbs allow for the removal of at least one target from plasma. Therefore, in some embodiments, the engineered BsAbs possess target-clearance activity against at least one target, wherein the at least one target is selected from TREM1 and interleukins.

[0245] In some embodiments, the BsAb described herein comprises an interleukin-binding region and a TREM1 (TREM1 and / or sTREM1)-binding region. In some embodiments, the interleukin-binding region comprises a first HC region, and the TREM1-binding region comprises a second HC region. In some embodiments, the first HC region comprises any combination of CDR-H or variants thereof described in Tables 2, 9, 16, and 23, 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 described in Tables 2, 9, 16, and 23, respectively. In some embodiments, the first HC region comprises any one or a variant of the VH sequence described in Tables 3, 10, 17, and 24, 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 described in Tables 3, 10, 17, and 24, respectively. In some embodiments, the second HC region comprises any combination of the CDR-H sequence described in Table 30.1 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 parental CDR-H sequence described in Table 30.1. In some embodiments, the second HC region comprises any of the VH sequences or variants thereof described in Table 31, wherein the variants contain 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% of the same amino acid sequence as the corresponding parental VH sequence described in Table 31.

[0246] In some embodiments, the BsAb described herein comprises an interleukin-binding region containing a first LC, a TREM1-binding region containing a second LC, or a combination thereof. In some embodiments, the first LC region comprises any one of the CDR-L or variants thereof described in Tables 4, 11, 18, and 25, 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-L sequence described in Tables 4, 11, 18, and 25, respectively. In some embodiments, the first LC region comprises any one of the VL sequences or variants thereof described in Tables 6, 13, 20, and 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-L sequence described in Tables 6, 13, 20, and 27, respectively. In some embodiments, the second LC region comprises any combination of the CDR-L or variants thereof described in Table 32.1, wherein the variants comprise 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% of the amino acid sequence identical to the corresponding parental CDR-L sequence described in Table 32.1. In some embodiments, the second LC region comprises any VL sequence or variants thereof described in Table 34, wherein the variants comprise 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% of the amino acid sequence identical to the corresponding parental CDR-L sequence described in Table 34.

[0247] In some embodiments, the bispecific antibody described herein comprises at least one constant region. In some embodiments, the bispecific antibody described herein comprises two constant regions. In some embodiments, the two constant regions are derived from the human IgG1 heavy 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 engineered to include a node, and the second constant region is engineered to include a pore. Thus, in some embodiments, the first constant region contains a mutation at position T366 according to EU numbering, and the second constant region contains a mutation at position Y407 according to EU numbering. In some embodiments, the first constant region contains a mutation at position T366 according to EU numbering, and the second constant region contains mutations at positions T366 and Y407 according to EU numbering. In some embodiments, the first constant region contains a mutation at position T366 according to EU numbering, and the second constant region contains mutations at positions T366, L368, and Y407 according to EU numbering. In some implementations, the first constant region contains the S354C mutation, the T366W mutation, or a combination thereof according to the EU number, and the second constant region contains the Y349C mutation, the T366S mutation, the Y407V mutation, or a combination thereof according to the EU number.

[0248] Optionally, in some embodiments, the bispecific antibody described herein comprises two constant regions derived from the human IgG1 heavy constant chain, wherein the two constant regions comprise a first constant region and a second constant region, wherein the first constant region contains at least two mutations and the second constant region contains at least one mutation. For example, in some embodiments, the first constant region contains at least two mutations selected from positions L351, F405, and Y407 according to EU numbers, and the second constant region contains at least one mutation selected from positions T366, K392, and T394 according to EU numbers. In some embodiments, the mutation at position L351 includes L351Y and L351A substitutions. In some embodiments, the mutation at position F405 includes F405A, F405S, F405T, and F405V substitutions. In some embodiments, the mutation at position Y407 includes Y407A, Y407V, Y407S, and Y407I substitutions. In some embodiments, the mutation at position T366 includes substitutions of T366L, T366M, T366V, and T366I. In some embodiments, the mutation at position K392 includes substitutions of K392C, K392M, K392L, K392I, K392E, K392D, and K392F. In some embodiments, the mutation at position T394 includes substitutions of T394D, T394W, T394V, and T394S. In some embodiments, at least two mutations in the first constant region also include one or more mutations at positions Q347, Y349, T350, K370, G371, D399, and S400 according to EU numbers. In some embodiments, the mutation at position Q347 includes substitutions of Q347R, Q347E, and Q347K. In some embodiments, the mutation at position Y349 includes a substitution of Y349C. In some embodiments, the mutation at position T350 includes a T350V substitution. In some embodiments, the mutation at position K370 includes a K370T substitution. In some embodiments, the mutation at position G371 includes G371D and G371S substitutions. In some embodiments, the mutation at position D399 includes D399C, D399R, and D399K substitutions. In some embodiments, the mutation at position S400 includes S400D, S400K, S400E, and S400R substitutions. In some embodiments, at least two mutations in the second constant region also include one or more mutations at positions T350, S354, E357, K360, Q362E, S364, N390, K409, and T411 according to EU numbers. In some embodiments, the mutation at position T350 includes a T350V substitution. In some embodiments, the mutation at position S354 includes an S354C substitution. In some implementations, the mutation at position E357 includes the E357Q substitution.In some embodiments, the mutation at position K360 includes K360D and K360E substitutions. In some embodiments, the mutation at position Q362 includes Q362E substitution. In some embodiments, the mutation at position S364 includes S364R substitution. In some embodiments, the mutation at position N390 includes N390K, N390R, N390D, and N390E substitutions. In some embodiments, the mutation at position K409 includes K409L, K409M, K409F, and K409W substitutions. In some embodiments, the mutation at position T411 includes T411R, T411D, T411I, T411K, T411E, T411N, T411S, and T411L substitutions.

[0249] Optionally, in some embodiments, the bispecific antibody described herein comprises two constant regions derived from the human IgG1 heavy constant chain, wherein the two constant regions comprise a first constant region and a second constant region, wherein the first and second constant regions are engineered to electrostatically interact with each other. In some embodiments, the first constant region comprises a substitution of a negatively charged amino acid residue (e.g., aspartic acid, glutamic acid) at K370 according to EU designation, and the second constant region comprises a substitution of a positively charged amino acid residue (e.g., arginine, lysine, histidine) at E357 according to EU designation. In some embodiments, the first constant region comprises a substitution of a negatively charged amino acid residue (e.g., aspartic acid, glutamic acid) at K392 or K409 according to EU designation, and the second constant region comprises a substitution of a positively charged amino acid residue (e.g., arginine, lysine, histidine) at D399 according to EU designation. In some embodiments, the first constant region comprises the substitution of a negatively charged amino acid residue (e.g., aspartic acid, glutamic acid) at K439 according to EU number, and the second constant region comprises the substitution of a positively charged amino acid residue (e.g., arginine, lysine, histidine) at D356 according to EU number.

[0250] In some implementations, treatment with the BsAb described herein reduces the level of at least one inflammatory marker (e.g., a biomarker) in a subject's serum by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% relative to the level of at least one inflammatory marker (e.g., a biomarker) after treatment with a corresponding combination of two monospecific antibodies. Therefore, treatment with a BsAb binding TREM1 and interleukins (e.g., the IL-1 family, IL-6 family, IL-12 family, IL-23 family) reduces the level of at least one biomarker in a subject's serum by 10% or more relative to the level of at least one biomarker after treatment with a corresponding combination of a TREM1-binding monospecific antibody and an interleukin-binding monospecific antibody. In some embodiments, at least one biomarker includes matrix metalloproteinase 1 (MMP1), matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 7 (MMP7), matrix metalloproteinase 10 (MMP10), granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor-α (TNFα), tumor necrosis factor superfamily member 15 (TNFSF15), IL-17, IL-1α, IL-1β, IL-6, IL-8, 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), chitosanase-3-like protein 1 (CHI3L1), prostaglandin-endoperoxidase 2 (PTGS2), and secretogranin V. (SCG5), inhibin β-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.

[0251] BiTE antibody In some embodiments, the molecules described herein comprise a bispecific T-cell adaptor (BiTE) antibody construct and may be referred to herein as "BiTE molecules". A 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 also comprises an interleukin-binding domain, wherein the interleukin-binding domain is capable of binding to a protein, a variant thereof, or a functional fragment thereof selected from any one of the IL-1 family, IL-6 family, IL-12 family, and IL-23 family.

[0252] Chemical crosslinking The covalent linking of two antibodies using chemical crosslinking agents is known in the art. Functional antibody fragments generated from their respective parent antibodies by enzymatic digestion or by recombinant technology can be conjugated using bifunctional agents (Glennie MJ et al., J Exp Med 1992; 175:217-225).

[0253] Quadromas Tetravalent and trivalent hybridomas can be generated by fusing two 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 leads to the random assembly of 10 antibody combinations, with the desired BsAbs comprising only a small fraction of the secreted antibodies. BsAbs can be purified using a combination of chromatographic techniques.

[0254] Recombinant multispecific antibodies Most multispecific antibody formats can be generated using genetic engineering techniques with functional antibody fragments such as scFv or Fab fragments as building blocks linked via peptide linkers. Linkage-based functional antibody fragment formats include tandem scFv (BiTE), biantibodies, and tandem biantibodies (Kipriyanov SM. MethodsMol Biol 2003; 207:323-333; Korn T et al., Int J Cancer 2002; 100:690-697). These formats include biantibody-Fc, tandem biantibody-Fc, tandem biantibody-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 a Fab2 antibody, a biscFv antibody, a biantibody, DVD-Ig, TandAb, tandem scFv-Fc, a single-arm tandem scFv-Fc, DART, DART-Fc, or a functional fragment thereof. Another type of multispecific antibody and its construction are disclosed in Brinkmann & Kontermann (2017), mAbs, 9:2, 182-212, DOI: 10.1080 / 19420862.2016.1268307, the contents of which are incorporated herein by reference.

[0255] Strategies based on forced heterodimerization of two heavy chains have been explored. The first approach, known as the “knobinto hole,” aims to force pairing of two distinct IgG heavy chains by introducing mutations into the CH3 domain to modify the contact interface (Ridgway JB et al., Protein Eng 1996; 9: 617-621). An amino acid with a large side chain is introduced onto one chain to create a “knob.” Conversely, the bulky amino acid is replaced with an amino acid with a short side chain to create a “hole” in the other CH3 domain. By co-expressing these two heavy chains, more than 90% of heterodimer formation (“hole-hole” or “knob-knob”) was observed relative to homodimer formation (“knob-hole”). A similar concept was developed using human CH3 domains with chain-exchange engineered domains (SEED) based on human IgG and human IgA sequences (Davis JH et al., 2010, PEDS 23: 195-202). These engineered domains lead to the formation of heterodimer molecules that can carry two different specificities.

[0256] The "joint-entry hole" method has recently been improved; "CrossMab" has been described in WO 2009 / 080253A1. In addition to the "joint-entry hole" mutation, the method also includes exchanging some light chain and heavy chain domains.

[0257] Antibodies based on single domains The immune systems of camels (lambs and camels) and cartilaginous fish (nurse sharks) use a single V domain fused to an Fc, demonstrating that a single domain can confer high affinity binding to antigens. The V domains in camels, sharks, and even humans represent alternatives to antibodies, but they can also be used for BsAb production. They can be reformulated into classic IgG, where each arm has the potential to bind to both targets via its VH or VL domain.

[0258] The bispecific antibodies disclosed herein can be prepared by any method disclosed in this application or known in the art.

[0259] Dual variable domain immunoglobulins An exemplary bispecific antibody may comprise a single peptide or "segment" encoden within a single continuous chain, containing a compact tertiary structure. The component peptides are selected to be structurally asymmetric so that they do not self-associate to form homodimers, but rather associate in a complementary manner, forming a stable complex similar to the parental tertiary structure. At the genetic level, these segments may be encoded by interchangeable cassettes with suitable restriction sites. These normalized cassettes can be fused to different recombinant proteins at the C- or N-terminus via linkers or hinges in a suitable expression vector system. Peptide segments lacking the ability to assemble into homodimers are derived by cleaving a parental peptide with a compact tertiary structure. These peptide segments can then be fused at the gene level to one or more distinct functional domains. These distinct peptide segments, now fused to one or more functional domains, can be co-expressed, for example, resulting in the formation of a native-like parental structure attached to the functional domains. This parental structure is formed through the dimerization of peptide segments derived from the original parental peptide. The resulting multifunctional construct will exhibit a compact tertiary structure attached to one or more functional domains. After the structural subdomains are identified, the protein is dissected in a manner that preserves the integrity of these subdomains. As part of this disclosure, DNA sequences, vectors, preferably bicistronic vectors, and vector cassettes can be prepared, characterized in that they comprise a DNA sequence encoding an amino acid sequence and at least one additional (poly)peptide optionally included in the multifunctional polypeptide of the present invention, and at least one additional, preferably single, cloning site for inserting DNA encoding at least one additional functional domain, or characterized in that they comprise a DNA sequence encoding an amino acid sequence and one or more additional (poly)peptides optionally included in the multifunctional polypeptide of the present invention and suitable restriction sites for cloning DNA sequences encoding functional domains, such that, after inserting the DNA sequence encoding the functional domains into said restriction sites, the multifunctional polypeptide of the present invention can be formed when the DNA sequence is expressed in a suitable host. The carrier cassette is characterized by comprising one or more insert DNA sequences encoding one or more of the said functional domains and a host cell transformed with at least one of the vectors or carrier cassettes of the present invention, which can be used to prepare the bispecific or multifunctional polypeptide. The host cell can be a mammal, preferably human, yeast, insect, plant, or bacterium, preferably *E. coli* cells. The bispecific antibody can be prepared by a method comprising culturing at least two types of host cells of the present invention in a suitable culture medium, each host cell producing only one of the first and second amino acid sequences attached to at least one additional functional domain, recovering the amino acid sequences, mixing them under mild denaturing conditions, and allowing the multifunctional polypeptide of the present invention to fold in vitro from the amino acid sequences.The method may be characterized in that the additional amino acid sequence attached to at least one additional functional domain is produced by at least one additional host cell that does not produce the first amino acid sequence or the second amino acid sequence. Alternatively, the method may be characterized in that the at least one additional amino acid sequence attached to at least one additional functional domain is produced by the host cell of the present invention that produces the first amino acid sequence or the second amino acid sequence.

[0260] When the second or first part of the antibody construct described herein contains two antibody variable domains, these two antibody variable domains can be VH and VL domains associated with each other. However, it is also contemplated that the two antibody variable domains contained in the second or first part can be two VH domains or two VL regions associated with each other. In the case where the two antibody variable domains in the first or second part are covalently associated with each other, the two antibody variable domains can be designed as scFv fragments, meaning that the two domains are separated from each other by a sufficiently long peptide linker to allow intermolecular association between the two domains. The design of linkers suitable for this purpose is described in the prior art, for example in Patent EP 623 679 B1, U.S. Patent No. 5,258,498, EP 573 551 B1, and U.S. Patent No. 5,525,491. In other words, a bispecific antibody can be a construct having a total of three antibody variable domains. Specifically, one antibody variable domain alone (i.e., not paired with another antibody variable domain) (a) specifically binds to human immune effector cells or target cells by specifically binding to effector antigens on human immune effector cells, while the remaining two antibody variable domains together specifically bind to (b) target antigens on target cells or specifically bind to human immune effector cells by specifically binding to effector antigens on human immune effector cells. In this case, the presence of three antibody variable domains in a bispecific antibody has unique advantages. Typically, scFvs exhibiting the desired binding specificity to the target antigen are known and optimized, and omitting one of their two antibody variable domains would eliminate or at least weaken their binding characteristics. Such scFvs can form part of the antibody constructs described herein. In particular, such tri-domain antibodies can advantageously contain the complete scFv as its effector antigen or target antigen-conferred portion. Thus, effectively, this allows bispecific antibodies to be formed from a desired scFv by simply incorporating only one additional antibody variable domain into the same polypeptide chain as the scFv, wherein the incorporated additional antibody variable domain has an antigen-binding specificity different from that of the scFv. The first and second portions of a bispecific antibody can be separated from each other by a synthetic polypeptide spacer that covalently links the C-terminus of the first portion to the N-terminus of the second portion (i.e., peptideically), or vice versa. Therefore, the portions of these bispecific antibodies can be arranged as 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-terminus or C-terminus of the heavy or light chain, for example, in the form of an scFv fragment or a variable single domain, to create a bispecific tetravalent molecule.Bispecific molecules generated by fusing scFv fragments with mAbs offer significant flexibility. scFv molecules can be linked to the N-terminus or C-terminus of the variable domains of either the heavy or light chain of the mAb, typically without compromising productivity or antigen-binding activity. This group of bispecific molecules also includes DVD-Ig (where the second VH and VL domains are fused to the heavy and light chains of the mAb (a dual-antibody), respectively, with the second specificity introduced into the native binding site of the IgG molecule), and mAb2 molecules (where the second specificity is constructed into the CH3 domain of the Fc region). All these molecules are characterized by symmetry resulting from the dimer assembly of two identical heavy chains, an inherent property of these chains.

[0261] Heavy chain heterodimerization can be achieved by engineering charged CH3 interfaces to introduce electrostatic directing effects or by using chain-exchange engineered domain technology (SEEDbody), where the CH3 sequence contains alternating segments from human IgA and IgG. These bispecific antibodies are divalent compared to bispecific IgG-like molecules, and their size is substantially the same as that of IgG. Recently, Fc heterodimerization has been applied to generate trivalent bispecific molecules (HA-TF Fc variants) that fuse VH and VL domains to the C-terminus of engineered heavy chains. Bispecific antibodies with molecular weights in the 50 kDa–100 kDa range can be generated by combining variable domains of two antibodies. For example, two scFvs have been linked in tandem orientation via more or less flexible peptide linkers (tandem scFv, taFv, tascFv), which can be further extended by additional scFvs, for example, to generate bispecific or trispecific trimers (sctb). Biantibodies are heterodimeric molecules containing variable domains of two antibodies arranged in a VHA-VLB and VHB-VLA sequence (VH-VL orientation) or a VLA-VHB and VLB-VHA sequence (VL-VH orientation). The linker connecting the two domains within a single chain is approximately 5 residues, which, upon co-expression of the two chains within a cell, leads to head-to-tail assembly, resulting in a compact molecule with two functional binding sites. The biantibody (Db) format is further stabilized by introducing interchain disulfide bonds (dsDb, DART molecules) or by generating single-chain derivatives (scDb). scDb can be converted to a tetravalent molecule by homodimerization of the two chains through reduction of the intermediate linker. Smaller bispecific molecules are also generated by fusing scFv with the heavy or light chain of a Fab fragment. Furthermore, tandem scFv, biantibody, and scDb have been fused with Fc or CH3 domains to generate tetravalent derivatives. Furthermore, scFv can be combined with Fc or CH3 domains to generate tetravalent molecules, for example, by fusing scFv with the N-terminus and C-terminus of an Fc fragment, or by using a pore-entry method to generate bivalent scFv-Fc or scFv-CH3 molecules. Different methods for generating the bispecific antibodies of the present invention are dock-and-lock (DNL) methods. Many established bispecific antibody formats can also be combined with additional proteins and components (e.g., drugs, toxins, enzymes, and cytokines) to achieve dual targeting and delivery of fusion couplers. Additionally, fusion with plasma proteins such as serum albumin or albumin-binding moieties can be applied to prolong the plasma half-life of bispecific antibodies.

[0262] Structure of bispecific antibodies In one example, the bispecific antibody may be a binding protein comprising a first polypeptide chain, wherein the polypeptide chain comprises VH1-(X1)n-VH2-C--(X2)n, where VH1 is a first heavy chain variable domain, VH2 is a second heavy chain variable domain, C is a constant domain, X1 represents a peptide 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-transplanted heavy chain variable domains, and humanized heavy chain variable domains. VH1 and VH2 may be able to bind 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 one embodiment, X2 is an Fc region. In another embodiment, X2 is a variant Fc region. In some embodiments, VH1 is capable of binding a first antigen and VH2 is capable of binding a second antigen. In some implementations, VH1 can bind to the second antigen and VH2 can bind to the first antigen.

[0263] In one example, the bispecific antibody may be a binding protein comprising a second polypeptide chain, wherein the polypeptide chain comprises VL1-(X1)n-VL2-C--(X2)n, where 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-transplanted light chain variable domains, and humanized light chain variable domains. VL1 and VL2 may be able to bind different antigens. C may be a light chain constant domain. For example, X1 is a linker peptide. For example, X1 is a linker listed herein. In one embodiment, X2 is an Fc region. In another embodiment, X2 is a variant Fc region. In some embodiments, VL1 is capable of binding a first antigen and VL2 is capable of binding a second antigen. In some embodiments, VL1 is capable of binding a first antigen and VL2 is capable of binding 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 dual variable domain immunoglobulin (DVD-Ig™), as described in Jakob 2013, which combines the target-binding domains of two monoclonal antibodies via a flexible, naturally occurring linker, resulting in a tetravalent IgG-like molecule.

[0264] This disclosure further provides a method for preparing DVD-Ig binding proteins by pre-selecting parental antibodies against a first antigen and a second antigen. A method for preparing a dual variable-domain immunoglobulin binding to two antigens includes the following steps: a) obtaining a first parental antibody or an antigen-binding fragment thereof that binds to a first antigen; b) obtaining a second parental antibody or an antigen-binding fragment thereof that binds to a second antigen; c) constructing two copies of a first polypeptide chain, each copy containing 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 fragment; VH2 is a second heavy-chain variable domain obtained from the second parental antibody or its antigen-binding fragment, the second parental antibody may be the same as or different from the first parental antibody; C is a heavy-chain constant domain; (X1)n is a linker, wherein (X1)n may or may not be present; and (X2)n is the Fc region; d) construct two copies of the second polypeptide chain, each copy containing 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 portion; VL2 is a second light chain variable domain obtained from the second parent antibody or its antigen-binding portion, the second parent antibody 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; and (X2)n does not contain the Fc region, wherein (X2)n may or may not be present; and e) express two copies of the first polypeptide chain and the second polypeptide chain, such that DVD-Ig binds to the first antigen and produces the second antigen.

[0265] Generates a first antigen-binding domain and / or a second antigen-binding domain This document discloses dual variable-domain immunoglobulins. The variable domains of DVD-binding proteins can be obtained from parental antibodies, including polyclonal antibodies and mAbs that bind to antigens of interest. These antibodies can be naturally occurring, generated via recombinant technologies, or designed de novo. mAbs can be prepared using a variety of techniques known in the art, including hybridoma, recombinant, and phage display technologies, or combinations thereof. Monoclonal antibodies can be prepared using the methods disclosed herein.

[0266] This document discloses a dual variable-domain immunoglobulin containing variable domains. The dual variable-domain immunoglobulin (DVD-Ig) molecule is engineered such that two distinct light chain variable domains (VLs) from two different parental monoclonal antibodies are tandemly linked directly or via short adapters using recombinant DNA technology, followed by a light chain constant domain and optionally an Fc region. Similarly, the heavy chain contains two distinct heavy chain variable domains (VHs) tandemly linked, followed by a constant domain CH1 and an Fc region. The variable domains can be obtained from parental antibodies produced using any of the methods described herein using recombinant DNA technology. The variable domains can be mouse heavy chain variable domains or light chain variable domains, CDRs, human heavy chain variable domains, or human light chain variable domains. The first and second variable domains can be directly linked to each other using recombinant DNA technology, linked via adapter sequences, or the two variable domains can be linked together. The variable domains can bind to the same antigen or can bind to different antigens. The constant domain can be linked to the two linked variable domains using recombinant DNA technology. A sequence containing a linker of the heavy chain variable domain can be linked to a heavy chain constant domain, and a sequence containing a linker of the light chain variable domain can be linked to a light chain constant domain. The constant domains can also be human heavy chain constant domains and human light chain constant domains, respectively. The DVD heavy chain can also be linked to an Fc region. The Fc region can be a native sequence Fc region, a variant Fc region, or a human Fc region. Two heavy chain DVD peptides and two light chain DVD peptides can combine to form a DVD-Ig molecule.

[0267] 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 comprises an IgG1 heavy constant chain. In some embodiments, the IgG1 heavy constant chain comprises a human IgG1 heavy constant chain. The amino acid sequence of the wild-type human IgG1 heavy constant chain is provided in Table 40. In some embodiments, the Fc region is engineered to not bind to an Fcγ receptor (FcγR). In some embodiments, the FcγR comprises FcγRI, FcγRII, and FcγRIII.

[0268] Table 40. Amino acid sequence of the Fc region of human IgG1

[0269] In some embodiments, the Fc region contains 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% of the amino acid sequence that is identical to the corresponding parental sequence (SEQ ID NO:453) of the Fc region of IgG1.

[0270] In some embodiments, the Fc region described herein contains one or more mutations relative to the parental sequence (SEQ ID NO: 453) 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 relative to the parental sequence of SEQ ID NO: 453. 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 relative to the parental sequence of SEQ ID NO: 453. In some implementations, 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 substitutions relative to the corresponding parental sequence of SEQ ID NO:453.

[0271] In some embodiments, the Fc region described herein is derived from the 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, the at least one substitution is selected from 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 according to EU designations. In some implementations, at least two substitutes are selected from 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 according to EU numbering. In some implementations, at least three substitutions are selected from 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 according to EU designations. In some implementations, at least four substitutions are selected from 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 according to EU designations. In some implementations, at least five substitutions are selected from 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 according to EU numbering.In some implementations, at least six substitutions are selected from 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 according to EU designations. In some embodiments, at least seven substitutions are selected from 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 according to EU designations. In some embodiments, the substitution at position N297 includes N297A and N297Q substitutions. In some embodiments, the substitution at position C226 includes C226S substitution. In some embodiments, the substitution at position C229 includes C229S substitution. In some embodiments, the substitution at position E233 includes E233P and E233D substitutions. In some embodiments, the substitution at position L234 includes substitutions of L234A, L234D, L234E, L234G, L234H, L234K, L234Q, L234R, L234S, L234T, and L234F. In some embodiments, the substitution at position L235 includes substitutions of L235A, L235S, L235T, L235H, L235K, L235Q, L235D, L235I, L235V, L235R, L235E, and L235G. In some embodiments, the substitution at position G236 includes a substitution of G236R. In some embodiments, the substitution at position G237 includes substitutions of G237A and G237D. In some embodiments, the substitution at position P238 includes substitutions of P238A, P238D, and P238S. In some embodiments, the substitution at position F243 includes a substitution of F243L. In some embodiments, the substitution at position M252 includes a substitution of M252Y. In some embodiments, the substitution at position S254 includes a substitution of S254T. In some embodiments, the substitution at position T256 includes a substitution of T256E. In some embodiments, the substitution at position D265 includes a substitution of D265A. In some embodiments, the substitution at position S267 includes a substitution of S267E. In some embodiments, the substitution at position H268 includes substitutions of H268A and H268D. In some embodiments, the substitution at position D270 includes a substitution of D270A.In some embodiments, the substitution at position P271 includes a P271G substitution. In some embodiments, the substitution at position R292 includes an R292P substitution. In some embodiments, the substitution at position Y300 includes a Y300L substitution. In some embodiments, the substitution at position K322 includes K322A and K322Q substitutions. In some embodiments, the substitution at position A327 includes A327Q and A327G substitutions. In some embodiments, the substitution at position L328 includes L328E and L328F substitutions. In some embodiments, the substitution at position P329 includes P329G and P329A substitutions. In some embodiments, the substitution at position A330 includes A330S, A330R, and A330L substitutions. In some embodiments, the substitution at position P331 includes a P331S substitution. In some embodiments, the substitution at position P396 includes a P396L substitution.

[0272] In some embodiments, the human IgG1 heavy chain constant chain described herein comprises two substitutions. In some embodiments, the two substitutions are located at positions L234 and L235 according to EU designations. In some embodiments, the two substitutions are L234A and L235A substitutions.

[0273] In some embodiments, the human IgG1 heavy chain constant chain described herein comprises three substitutions. In some embodiments, the three substitutions are located at positions L234, L235, and P329 according to EU numbers. 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 according to EU numbers. In some embodiments, the three substitutions are M252T, S254T, and T256E substitutions.

[0274] In some embodiments, the Fc region contains 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% of the amino acid sequence that is identical to the corresponding parental sequence (SEQ ID NO:454) of the Fc region of IgG2.

[0275] In some embodiments, the Fc region described herein contains one or more mutations relative to the parental sequence (SEQ ID NO: 454) corresponding to 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 relative to the parental sequence of SEQ ID NO: 454. 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 relative to the parental sequence of SEQ ID NO: 454. In some implementations, 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 substitutions relative to the corresponding parental sequence of SEQ ID NO:454.

[0276] In some embodiments, the Fc region described herein is derived from the human IgG2 heavy chain constant. In some embodiments, the human IgG2 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 positions C232, C233, V234, G237, P238, M252, S254, T256, H268, N297, V309, A330, and P331 according to EU numbers. In some embodiments, at least two substitutions are selected from positions C232, C233, V234, G237, P238, M252, S254, T256, H268, N297, V309, A330, and P331 according to EU numbers. In some embodiments, at least three substitutions are selected from positions C232, C233, V234, G237, P238, M252, S254, T256, H268, N297, V309, A330, and P331 according to EU designations. In some embodiments, at least four substitutions are selected from positions C232, C233, V234, G237, P238, M252, S254, T256, H268, N297, V309, A330, and P331 according to EU designations. In some embodiments, at least five substitutions are selected from positions C232, C233, V234, G237, P238, M252, S254, T256, H268, N297, V309, A330, and P331 according to EU designations. In some embodiments, at least six substitutions are selected from positions C232, C233, V234, G237, P238, M252, S254, T256, H268, N297, V309, A330, and P331 according to EU designations. In some embodiments, at least seven substitutions are selected from positions C232, C233, V234, G237, P238, M252, S254, T256, H268, N297, V309, A330, and P331 according to EU designations. In some embodiments, the substitution at position C232 includes a C232S substitution. In some embodiments, the substitution at position C233 includes a C233S substitution. In some embodiments, the substitution at position V234 includes a V234A substitution. In some embodiments, the substitution at position G237 includes a G237A substitution. In some embodiments, the substitution at position P238 includes a P238S substitution. In some embodiments, the substitution at position M252 includes an M252Y substitution. In some embodiments, the substitution at position S254 includes an S254T substitution. In some embodiments, the substitution at position T256 includes a T256E substitution.In some embodiments, the substitution at position H268 includes substitutions of H268A, H268E, and H268Q. In some embodiments, the substitution at position N297 includes substitutions of N297A and N297Q. In some embodiments, the substitution at position V309 includes substitution of V309L. In some embodiments, the substitution at position A330 includes substitution of A330S. In some embodiments, the substitution at position P331 includes substitution of P331S.

[0277] In some embodiments, the Fc region contains 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% of the amino acid sequence that is identical to the corresponding parental sequence (SEQ ID NO:455) of the Fc region of IgG4.

[0278] In some embodiments, the Fc region described herein contains one or more mutations relative to the parental sequence (SEQ ID NO: 455) 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 relative to the parental sequence of SEQ ID NO: 455. 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 relative to the parental sequence of SEQ ID NO: 455. In some implementations, 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 substitutions relative to the corresponding parental sequence of SEQ ID NO:455.

[0279] In some embodiments, the Fc region described herein is derived from the 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, the at least one substitution is selected from positions S228, E233, F234, L235, L236, G237, S241, L248, M252, S254, T256, N297, E318, and T394 according to EU numbers. In some embodiments, the at least two substitutions are selected from positions S228, E233, F234, L235, L236, G237, S241, L248, M252, S254, T256, N297, E318, and T394 according to EU numbers. In some embodiments, at least three substitutions are selected from positions S228, E233, F234, L235, L236, G237, S241, L248, M252, S254, T256, N297, E318, and T394 according to EU designations. In some embodiments, at least four substitutions are selected from positions S228, E233, F234, L235, L236, G237, S241, L248, M252, S254, T256, N297, E318, and T394 according to EU designations. In some embodiments, at least five substitutions are selected from positions S228, E233, F234, L235, L236, G237, S241, L248, M252, S254, T256, N297, E318, and T394 according to EU designations. In some embodiments, at least six substitutions are selected from positions S228, E233, F234, L235, L236, G237, S241, L248, M252, S254, T256, N297, E318, and T394 according to EU designations. In some embodiments, at least seven substitutions are selected from positions S228, E233, F234, L235, L236, G237, S241, L248, M252, S254, T256, N297, E318, and T394 according to EU designations. In some embodiments, the substitution at position S228 includes the S228P substitution. In some embodiments, the substitution at position E233 includes the E233P substitution. In some embodiments, the substitution at position F234 includes the F234V substitution. In some embodiments, the substitution at position L235 includes the L235A substitution. In some embodiments, the substitution at position G237 includes a G237A substitution. In some embodiments, the substitution at position S241 includes an S241P substitution. In some embodiments, the substitution at position L248 includes an L248E substitution.In some embodiments, the substitution at position M252 includes an M252Y substitution. In some embodiments, the substitution at position S254 includes an S254T substitution. In some embodiments, the substitution at position T256 includes a T256E substitution. In some embodiments, the substitution at position N297 includes N297A and N297Q substitutions. In some embodiments, the substitution at position E318 includes an E318A substitution. In some embodiments, the substitution at position T394 includes a T394D substitution.

[0280] The design of the “dual-specific multivalent full-length binding protein” disclosed herein generates a dual-variable domain light chain and a dual-variable domain heavy chain that are primarily assembled into the desired “dual-specific multivalent full-length binding protein”.

[0281] Construction of DVD molecules This article discloses a dual variable domain immunoglobulin. The dual variable domain immunoglobulin (DVD-Ig) molecule is engineered such that two distinct light chain variable domains (VL) from two parental monoclonal antibodies (which may be identical or different) are tandemly linked directly or via short adapters using recombinant DNA technology, followed by a light chain constant domain and optionally an Fc region. Similarly, the heavy chain comprises two distinct heavy chain variable domains (VH) tandemly linked, followed by a constant domain CH1 and an Fc region.

[0282] This document discloses a dual variable-domain immunoglobulin comprising a variable domain. The variable domain can be obtained from a parental antibody produced using recombinant DNA technology through any of the methods described herein. In one embodiment, the variable domain is a mouse heavy chain variable domain or a mouse light chain variable domain. In another embodiment, the variable domain is a CDR-transplanted or humanized variable heavy chain or light chain domain. In one embodiment, the variable domain is a human heavy chain variable domain or a human light chain variable domain.

[0283] This document discloses a dual-variable-domain immunoglobulin comprising a first variable domain and a second variable domain. 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 adapter sequences. In some embodiments, the two variable domains are linked. Three or more variable domains may also be directly linked or linked via adapter sequences. Variable domains may bind to the same antigen or may bind to different antigens. The DVD-Ig molecule of this invention may comprise an immunoglobulin variable domain and a 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.

[0284] In one embodiment, the constant domain is linked to two linked variable domains using recombinant DNA technology. In this embodiment, a sequence containing the linked heavy chain variable domain is linked to the heavy chain constant domain, and a sequence containing the linked light chain variable domain is linked to the light chain constant domain. In this embodiment, the constant domains are the human heavy chain constant domain and the human light chain constant domain, respectively. The DVD-Ig molecule described herein may contain the DVD heavy chain. In this embodiment, the DVD heavy chain is also linked to an Fc region. The Fc region may be a natural sequence Fc region or a variant Fc region. In another embodiment, the Fc region is human.

[0285] In another embodiment, two heavy-chain DVD peptides and two light-chain DVD peptides are combined to form a DVD-Ig molecule.

[0286] The binding protein of the present invention can be produced by any of a variety of techniques known in the art. For example, it can be expressed from host cells, wherein one or more expression vectors encoding the DVD heavy chain and the DVD light chain are transfected into the host cells using standard techniques. Various forms of the term "transfection" are intended to encompass a variety of techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, etc. Although it is possible to express the DVD protein of the present invention in prokaryotic or eukaryotic host cells, the DVD protein is expressed in eukaryotic cells, such as mammalian host cells, because such eukaryotic cells (and especially mammalian cells) are more likely than prokaryotic cells to assemble and secrete properly folded and immunologically active DVD proteins.

[0287] Exemplary mammalian host cells used for expressing the recombinant antibodies of the present invention include Chinese hamster ovary (CHO) cells (including dhfr-CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA77:4216-4220, used with DHFR selection markers, e.g., as described in Kaufman, RJ and Sharp, PA (1982) Mol. Biol. 159:601-621), NSO myeloid cells, COS cells, SP2 and PER.C6 cells. When a recombinant expression vector encoding the DVD protein is introduced into mammalian host cells, the DVD protein is produced by culturing the host cells for a period of time sufficient to allow the DVD protein to be expressed in the host cells or secreted into the culture medium in which the host cells grow. The DVD protein can be recovered from the culture medium using standard protein purification methods.

[0288] In an 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 via calcium phosphate-mediated transfection. Within the recombinant expression vector, the DVD heavy chain and light chain genes are each operatively linked to a CMV enhancer / AdMLP promoter regulatory element to drive high-level transcription of the genes. The recombinant expression vector also carries a DHFR gene that allows selection of CHO cells already transfected with the vector using methotrexate selection / amplification. The selected transformant host cells are cultured to allow expression of the DVD heavy chain and light chain, and the 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 the DVD protein from the culture medium. Furthermore, the present invention also 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. This method may further include isolating the DVD protein from the culture medium.

[0289] A key characteristic of DVD-Ig is its ability to be generated and purified in a manner similar to conventional antibodies. DVD-Ig production results in a homogeneous, single major product with the desired dual-specific activity, without any sequence modifications to the constant region or any kind of chemical modification. Other previously described methods for generating “bispecific,” “multispecific,” and “multispecific multivalent” full-length binding proteins do not result in a single primary product, but rather in the intracellular or secretory production of a mixture of assembled inactive, single-specific, multispecific, multivalent, full-length binding proteins, and multivalent full-length binding proteins with different combinations of binding sites. 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. Therefore, compared to the other 15 possible combinations, only 6.25% of the protein is likely to be the desired active form, rather than as a single major product or a single primary product. The separation of the desired fully active form of protein from inactive and partially active forms using standard chromatographic techniques commonly used for large-scale production remains to be confirmed.

[0290] The design for the “dual-specific, multivalent, full-length binding protein” used in the constructs described herein produces a dual-variable-domain light chain and a dual-variable-domain heavy chain that are primarily assembled into the desired “dual-specific, multivalent, full-length binding protein”.

[0291] In some embodiments, at least 50%, at least 75%, and at least 90% of the assembled and expressed dual-variable-domain immunoglobulin molecules are desired dual-specific tetravalent proteins. This aspect of the invention particularly enhances its commercial applicability. Therefore, the invention includes a method for expressing a dual-variable-domain light chain and a dual-variable-domain heavy chain in a single cell, resulting in a single primary product of a “dual-specific tetravalent full-length binding protein.”

[0292] This article provides a method for expressing the dual variable domain light chain and dual variable domain heavy chain in single cells to produce a "primary product" of a "dual specific tetravalent full-length binding protein", wherein the "primary product" is more than 50% of the assembled protein containing both the dual variable domain light chain and the dual variable domain heavy chain.

[0293] This article provides a method for expressing a single “primary product” of a dual-variable domain light chain and a dual-variable domain heavy chain in a single cell, resulting in a “dual-specific tetravalent full-length binding protein”, wherein the “primary product” is more than 75% of the assembled protein containing both the dual-variable domain light chain and the dual-variable domain heavy chain.

[0294] This article provides a method for expressing a single “primary product” of a dual-variable domain light chain and a dual-variable domain heavy chain in a single cell, resulting in a “dual-specific tetravalent full-length binding protein”, wherein the “primary product” is more than 90% of the assembled protein containing both the dual-variable domain light chain and the dual-variable domain heavy chain.

[0295] Combining affinity Binding affinity is usually determined by the dissociation constant (K). D () represents the K of the antibody. D The value can be determined by any method known in the art. Used to determine K D Exemplary methods include the use of surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), spectrometry, biolayer interferometry (BLI), and grating-coupled interferometry (GCI).

[0296] In some embodiments, the multispecific antibodies described herein contain binding affinities in the following ranges: 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, 10 nM to 500 nM, 1 pM to 100 nM, 10 pM to 100 nM, 10 nM to 100 nM, 1 pM to 10 nM, 10 pM to 10 nM, 100 pM to 10 nM, 1 nM to 10 nM, 1 pM to 10 nM, 1 pM to 10 nM, 1 pM to 10 nM, 1 pM to 10 nM, 1 pM to 10 nM, 1 pM to 10 nM, 1 pM to 10 nM, 1 pM to 10 nM, 1 pM to 10 nM, 1 pM to 10 nM, 1 pM to 10 nM, 1 pM to 10 nM, 1 pM to 1 nM, 10 pM to 1 nM, 100 pM to 1 nM, 1 pM to 100 pM or 10 pM to 100 pM.

[0297] In some embodiments, the BsAb antibodies described herein contain average binding affinity to the target antigen in the following ranges: 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, 10 nM to 500 nM, 1 pM to 100 nM, 10 pM to 100 nM, 10 nM 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 10 nM, 1 nM to 10 nM, 1 pM to 1 nM, 10 pM to 1 nM, 100 pM to 1 nM, 1 pM to 100 pM or 10 pM to 100 pM.

[0298] In some embodiments, the multispecific antibody described herein comprises 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 is an interleukin binding domain. In some embodiments, the binding affinity of the multispecific antibody is measured using only one target molecule (e.g., TREM1, sTREM1, or an interleukin (e.g., any one of proteins from the IL-1, IL-6, IL-12, and IL-23 families)).

[0299] In some embodiments, the multispecific antibody described herein comprises a TREM1 binding region and an interleukin binding region. In some embodiments, the interleukin binding region binds interleukins, wherein interleukins include proteins from the IL-1 family, IL-6 family, IL-12 family, and IL-23 family. In some embodiments, the TREM1 binding region has a lower binding affinity for TREM1 than the interleukin binding region has for interleukin. In some embodiments, the multispecific antibody comprises a TREM1 binding region with a binding affinity for TREM1 that is at least two, three, four, five, ten, fifteen, twenty, forty, sixty, eighty, or one hundred times higher than the binding affinity of the interleukin binding region for interleukin. Optionally, in some embodiments, the interleukin binding region has a lower binding affinity for interleukin than the binding affinity of the TREM1 binding region for TREM1. Therefore, in some embodiments, the multispecific antibody includes an interleukin binding region with an interleukin binding affinity at least two, three, four, five, ten, fifteen, twenty, forty, sixty, eighty, or one hundred times higher than that of the TREM1 binding region to TREM1.

[0300] In some embodiments, the multispecific antibody described herein comprises a TREM1 binding region and an interleukin-binding region that binds to interleukins (e.g., proteins from the IL-1, IL-6, IL-12, and IL-23 families). In some embodiments, the interleukin-binding region includes an IL-1 binding region that binds to interleukins from the IL-1 family, an IL-6 binding region that binds to interleukins from the IL-6 family, an IL-12 binding region that binds to interleukins from the IL-12 family, an IL-23 binding region that binds to interleukins from the IL-23 family, or a combination thereof. In some embodiments, the TREM1 binding region has a lower binding affinity for sTREM1 than the interleukin-binding region has for interleukins in general. In some embodiments, the binding affinity of the TREM1 binding region to sTREM1 is at least two times, at least three times, at least four times, at least five times, at least ten times, at least fifteen times, at least twenty times, at least forty times, at least sixty times, at least eight times, or at least one hundred times higher than the binding affinity of the interleukin binding region to interleukin. Alternatively, in some embodiments, the binding affinity of the interleukin binding region to interleukin is lower than the binding affinity of the TREM1 binding region to sTREM1. Therefore, in some embodiments, the binding affinity of the interleukin binding region to interleukin is at least two times, at least three times, at least four times, at least five times, at least ten times, at least fifteen times, at least twenty times, at least forty times, at least sixty times, at least eight times, or at least one hundred times higher than the binding affinity of the TREM1 binding region to sTREM1.

[0301] Optionally, in some embodiments, the multispecific antibodies described herein undergo a co-binding event. For example, in some embodiments, the multispecific antibody undergoes a positive co-binding event, wherein binding of the multispecific antibody to the first target molecule results in an increased affinity for the second target molecule. For example, in some embodiments, the binding affinity of the TREM1-bound multispecific antibody to interleukin is lower than that of the TREM1-unbound multispecific antibody to interleukin. In some embodiments, the binding affinity of the interleukin-bound multispecific antibody to TREM1 is lower than that of the TREM1-unbound multispecific antibody to TREM1. Optionally, in some embodiments, the multispecific antibody undergoes a negative co-binding event, wherein binding of the multispecific antibody to the first target molecule results in a decreased affinity for the second target molecule. For example, in some embodiments, the binding affinity of the TREM1-bound multispecific antibody to interleukin is higher than that of the TREM1-unbound multispecific antibody to interleukin. In some embodiments, the binding affinity of a multispecific antibody bound to interleukin to TREM1 is higher than that of a multispecific antibody not bound to interleukin to TREM1. In some embodiments, the binding affinity of a multispecific antibody bound to sTREM1 to interleukin is lower than that of a multispecific antibody not bound to sTREM1 to interleukin. In some embodiments, the binding affinity of a multispecific antibody bound to interleukin to sTREM1 is lower than that of a multispecific antibody not bound to interleukin to sTREM1. Optionally, in some embodiments, the multispecific antibody undergoes a negative co-binding event, wherein binding of the multispecific antibody to the first target molecule results in a reduced binding affinity to the second target molecule. For example, in some embodiments, the binding affinity of a multispecific antibody bound to sTREM1 to interleukin is higher than that of a multispecific antibody not bound to sTREM1 to interleukin. In some implementations, the binding affinity of interleukin-bound multispecific antibodies to sTREM1 is higher than that of interleukin-unbound multispecific antibodies to sTREM1.

[0302] κ-λ antibody This article presents multispecific (e.g., bispecific, trispecific) antibodies in the κ-λ antibody format. The bispecific antibodies presented here share a common heavy chain and two light chains (one Kappa (K) and one Lambda (λ)), each with different specificities (i.e., two light chains, two specificities). The methods presented here generate molecules with specific binding, where diversity is confined to the VL region. These methods generate bispecific antibodies through controlled co-expression and purification of three chains (one VH chain and two VL chains).

[0303] This type of molecule contains two copies of a unique heavy-chain polypeptide, with a first light-chain variable region fused to a constant κ domain and a second light-chain variable region fused to a constant λ domain. Each combination site exhibits distinct antigen specificities for which both the heavy and light chains contribute. The light-chain variable region can be of the λ or κ family and is preferably fused to the λ and κ constant domains, respectively. This is preferred to avoid generating unnatural polypeptide linkages. However, it is also possible to obtain the bispecific antibody of the present invention by fusing the κ light-chain variable domain to the constant λ domain for the first specificity and fusing the λ light-chain variable domain to the constant κ domain for the second specificity.

[0304] The basic steps of an exemplary method are to identify two antibody Fv regions (each containing a variable light chain and a variable heavy chain domain) that have different antigen specificities and share the same heavy chain variable domain. Many methods for generating monoclonal antibodies and their functional fragments have been described. (See, for example, *Antibodies: A Laboratory Manual*, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference). A fully human antibody is an antibody molecule in which both the light and heavy chain sequences (including CDRs 1 and 2) are derived from human genes. The CDR 3 region may be of human origin or designed synthetically. Such antibodies are referred to herein as “human antibodies” or “fully human antibodies.” Human monoclonal antibodies can be prepared 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 utilized, and they can be produced using human hybridomas (see Cole et al., 1983. Proc Natl Acad Sci USA 80:2026-2030) or by transforming human B cells with EBV in vitro (see Cole et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).

[0305] Monoclonal antibodies can be generated, for example, by immunizing animals with a target antigen or an immunogenic fragment, derivative, or variant thereof. Alternatively, animals may be 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 generating xenogeneic 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 hereby incorporated by reference in their entirety.

[0306] Alternatively, antibodies can be obtained by screening libraries containing antibody or antigen-binding domain sequences that bind to the target antigen. Such libraries can be prepared, for example, in bacterial phages as fusions of bacterial phage coat proteins expressed on the surface of assembled phage particles and proteins or peptides encoding DNA sequences contained within the phage particles (i.e., "phage display libraries").

[0307] The reactivity of hybridomas generated from myeloma / B-cell fusions to the target antigen is then screened. Monoclonal antibodies can be prepared, for example, using hybridoma methods, such as those described in Kohler and Milstein, Nature, 256:495 (1975). In hybridoma methods, mice, hamsters, or other suitable host animals are typically immunized with an immunizing agent to induce the production or ability to produce lymphocytes that will specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro.

[0308] κ-λ antibodies with the same heavy chain variable domain can be generated using an antibody library in which the heavy chain variable domain is identical for all library members, and therefore diversity is limited to the light chain variable domain. Such libraries are described, for example, in WO 2010 / 135558. However, since the light chain variable domain is expressed together with the heavy chain variable domain, both domains contribute to antigen binding. To further facilitate this process, antibody libraries containing the same heavy chain variable domain and multiple λ or κ variable light chains can be used in parallel for in vitro selection of antibodies against different antigens. This method is capable of identifying two antibodies with a common heavy chain but one carrying a λ light chain variable domain and the other carrying a κ light chain variable domain, which can be used as building blocks for generating bispecific antibodies in the whole immunoglobulin format of the present invention. Many methods for modifying the Fc portion have been described and are applicable to the antibodies of the present invention (see, for example, Strohl, WR Curr Opin Biotechnol 2009 (6):685-91).

[0309] Another step in the exemplary implementation is to optimize the co-expression of the common heavy chain and the two different light chains in a single cell to allow assembly of the bispecific antibody of the present invention. If all polypeptides are expressed at the same level and assembled equally well to form immunoglobulin molecules, the ratio of monospecific (same light chain) to bispecific (two different light chains) should be 50%.

[0310] Co-expression of the heavy chain and two light chains yields a mixture of three different antibodies in the cell culture supernatant: two monospecific bivalent antibodies and one bispecific bivalent antibody. The latter must be purified from the mixture to obtain the molecule of interest. The method described herein greatly facilitates this purification procedure by using affinity chromatography media that specifically interact with the constant domains of the κ or λ light chain, such as CaptureSelectFab κ and CaptureSelectFab λ affinity matrices (BAC BV, Holland). This multi-step affinity chromatography purification method is efficient and generally applicable to the antibodies of this invention. This contrasts sharply with the need for specific purification methods to be developed and optimized for each bispecific antibody derived from tetravalent hybridomas or other cell lines expressing a mixture of antibodies. In fact, if the different antibodies in the mixture have similar biochemical characteristics, separating them using standard chromatographic techniques such as ion exchange chromatography can be challenging or simply impossible.

[0311] In some embodiments, the purified bispecific antibodies described herein are characterized as follows. Flow buffers and eluates from each affinity purification step can be analyzed by SDS-PAGE. The specificity and affinity of the κλ antibody can be determined by ELISA and surface plasmon resonance. The method of the present invention allows for the identification of antibodies with affinities in the sub-nanomolar to nanomolar range without optimization. This is not apparent because the diversity in the antibody libraries described herein is limited to light chains that contribute little to the binding energy of standard antibodies.

[0312] To avoid the requirement of obtaining two antibodies with κ and λ-type light chain variable domains, which are considered a limitation of the invention, the methods described herein allow for the production of hybrid light chains, wherein the λ variable domain can be fused with the κ constant domain, and conversely, the κ variable domain can be fused with the λ constant domain. In some embodiments, the methods for producing bispecific and / or multispecific antibodies use a completely serum-free, chemically defined process. These methods incorporate the most widely used mammalian cell line in the pharmaceutical industry, namely the Chinese hamster ovary (CHO) cell line. The methods described herein are used to produce both semi-stable and stable cell lines. The methods can be used to prepare the bispecific and / or multispecific antibodies of the invention on a small scale (e.g., in Erlenmeyer flasks) and a medium scale (e.g., in 25 L Wave bags). The methods are also readily adaptable to the large-scale production of the bispecific and / or multispecific antibodies of the invention, as well as antibody mixtures.

[0313] Treatment In some embodiments, this document discloses a method of treating a subject with appropriate need, the method comprising administering to the subject an effective amount of one or more engineered protein molecules described herein. In some embodiments, the engineered protein molecule comprises the TREM1-binding engineered protein molecule described herein. In some embodiments, the engineered protein molecule comprises the interleukin-binding engineered protein molecule described herein. In some embodiments, the engineered protein molecule comprises both the TREM1-binding engineered protein molecule and the interleukin-binding engineered protein molecule described herein. In some embodiments, the interleukin-binding engineered protein molecule is selected from IL-1-binding engineered protein molecules, IL-6-binding engineered protein molecules, IL-12-binding engineered protein molecules, IL-23-binding engineered protein molecules, or combinations thereof.

[0314] In some embodiments, this document discloses methods for treating a subject in need, comprising administering to the subject an effective dose of one or more engineered protein molecules described herein. In some embodiments, this document discloses methods for treating a subject in need, comprising administering to the subject an effective dose of a multispecific (e.g., bispecific, trispecific) antibody or multispecific (e.g., bispecific, trispecific) molecule or a pharmaceutical composition comprising the multispecific (e.g., bispecific, trispecific) antibody or multispecific (e.g., bispecific, trispecific) molecule described herein. In some embodiments, the subject suffers from any of the following conditions selected from the group consisting of: dementia, frontotemporal dementia, Alzheimer's disease, vascular dementia, mixed dementia, Kreutzfeldt-Jacob disease, normal pressure hydrocephalus, amyotrophic lateral sclerosis, Huntington's disease, taupathy. Diseases including Nasu-Hakola disease, stroke, acute trauma, chronic trauma, cognitive impairment, memory loss, lupus, acute and chronic colitis, rheumatoid arthritis, atherosclerosis, wound healing, Crohn's disease, inflammatory bowel disease, ulcerative colitis, obesity, essential tremor, central nervous system lupus, Behcet's disease, Parkinson's disease, Lewy body dementia, multiple system atrophy, Shy-Drager syndrome, progressive supranuclear palsy, corticobasal degeneration, acute disseminated encephalomyelitis, granulomatous disorders, sarcoidosis, age-related diseases, seizures, spinal cord injury, traumatic brain injury, age-related macular degeneration, glaucoma, retinitis pigmentosa, retinitis pigmentosa, lupus, arthritis, multiple sclerosis, low bone mineral density, osteoporosis, osteogenesis imperfecta, osteopetrotic disease, and Paget's disease of bone. In some implementation schemes, subjects had autoimmune conditions. In some implementations, 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 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, pulmonary hemorrhage nephritis syndrome, chronic inflammatory demyelinating polyneuropathy, allergies, and asthma. In some implementations, the subject has age-related Alzheimer's disease. In some implementations, the subject has a disease or condition selected from the group consisting of infectious diseases or autoimmune diseases.In some implementation schemes, the subjects had: rheumatoid arthritis, juvenile arthritis, psoriatic arthritis, ankylosing spondylitis, axial spondyloarthritis, psoriasis, hidradenitis suppurativa, ulcerative colitis, Crohn's disease, necrotizing enterocolitis, sepsis, or multiple sclerosis.

[0315] Inflammatory diseases or conditions are associated with increased activity and / or expression of TREM-1, interleukins described herein, or combinations thereof in subjects compared to subjects without inflammatory diseases or conditions (or before their development). Additionally, inflammatory diseases or conditions are also associated with increased activity or expression of one or more downstream inflammatory signaling proteins of TREM1, interleukins described herein, or combinations thereof in subjects compared to subjects without inflammatory diseases or conditions (or before their development). In some embodiments, one or more inflammatory conditions include dementia, frontotemporal dementia, Alzheimer's disease, vascular dementia, mixed dementia, Crout-Felter-Jacob disease, normal pressure hydrocephalus, amyotrophic lateral sclerosis, Huntington's disease, Tau proteinosis, Nasu-Hakola disease, stroke, acute trauma, chronic trauma, cognitive impairment, memory loss, lupus, acute and chronic colitis, rheumatoid arthritis, atherosclerosis, wound healing, Crohn's disease, inflammatory bowel disease, ulcerative colitis, obesity, and other conditions. Inflammatory tremor, central nervous system lupus, Behcet's disease, Parkinson's disease, Lewy body dementia, multiple system atrophy, Shy-Drager syndrome, progressive supranuclear palsy, corticobasal degeneration, acute disseminated encephalomyelitis, granulomatous disorders, sarcoidosis, age-related diseases, seizures, spinal cord injury, traumatic brain injury, age-related macular degeneration, glaucoma, retinitis pigmentosa, retinitis pigmentosa, lupus, arthritis, multiple sclerosis, low bone mineral density, osteoporosis, osteogenesis imperfecta, osteoporosis, and Paget's disease. In some implementations, 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, pulmonary hemorrhage nephritis syndrome, chronic inflammatory demyelinating polyneuropathy, allergies, and asthma. In some embodiments, one or more inflammatory conditions include inflammatory conditions of 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 infectious diseases 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 hidradenitis suppurativa.In some embodiments, one or more inflammatory conditions include ulcerative colitis, Crohn's disease, necrotizing enterocolitis, sepsis, and / or multiple sclerosis. Therefore, in some embodiments, a method of treating a subject's inflammatory disease or condition includes administering the BsAb described herein, wherein, relative to a subject treated with a monospecific antibody targeting TREM1 or an interleukin described herein, the BsAb more significantly reduces the activity and / or expression of TREM1, the interleukin described herein, one or more of their downstream signaling proteins, or combinations thereof. In some embodiments, a method of treating a subject's inflammatory disease or condition includes administering the BsAb described herein, wherein, relative to a subject treated with a combination of two monospecific antibodies, the BsAb more significantly reduces the activity and / or expression of TREM1, the interleukin described herein, one or more of their downstream signaling proteins, or combinations thereof, wherein the first monospecific antibody targets TREM1 and the second monospecific antibody targets an interleukin described herein.

[0316] In some embodiments, a multispecific (e.g., bispecific, trispecific) antibody or multispecific (e.g., bispecific, trispecific) molecule is administered together with one or more additional therapeutic agents. In some embodiments, a multispecific (e.g., bispecific, trispecific) antibody or multispecific (e.g., bispecific, trispecific) molecule and one or more additional therapeutic agents are administered co-administered. In some embodiments, a multispecific (e.g., bispecific, trispecific) antibody or multispecific (e.g., bispecific, trispecific) molecule and one or more additional therapeutic agents are administered sequentially.

[0317] Diagnostic methods In some embodiments, this document discloses a method for diagnosing a subject's condition, the method comprising incubating a sample with an effective amount of a composition comprising one or more engineered protein molecules described herein. In some embodiments, this document discloses a method for diagnosing a subject's condition, the method comprising incubating a sample with an effective amount of a composition comprising a multispecific (e.g., bispecific, trispecific) antibody or multispecific (e.g., bispecific, trispecific) molecule described herein. In some embodiments, the sample comprises tissue, blood, serum, plasma, saliva, urine, or a combination thereof. In some embodiments, the diagnosis is based on TREM1 expression levels, wherein an elevated level of sTREM1 in a subject relative to TREM1 levels in a healthy individual indicates that the individual has an autoimmune condition. In some embodiments, the diagnosis is based on TREM1 expression levels, wherein an elevated level of TREM1 in a subject relative to TREM1 levels in a subject prior to the development of an autoimmune condition. In some embodiments, the diagnosis is based on sTREM1 expression levels, wherein an elevated level of sTREM1 in a subject relative to sTREM1 levels in a healthy individual indicates that the individual has an infectious disease or an autoimmune condition. In some implementations, 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 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, pulmonary hemorrhage nephritis syndrome, chronic inflammatory demyelinating polyneuropathy, allergies, and asthma. In some implementations, autoimmune conditions include rheumatoid arthritis, juvenile arthritis, psoriatic arthritis, ankylosing spondylitis, axial spondyloarthritis, psoriasis, hidradenitis suppurativa, ulcerative colitis, Crohn's disease, necrotizing enterocolitis, sepsis, multiple sclerosis, or combinations thereof.

[0318] Composition This document discloses compositions comprising one or more engineered protein constructs described herein. In some embodiments, the engineered protein construct comprises the TREM1 binding moiety described herein. In some embodiments, the engineered protein construct comprises the interleukin binding moiety described herein. In some embodiments, the engineered protein molecule comprises both the TREM1 binding moiety and the interleukin binding moiety described herein. In some embodiments, the interleukin binding moiety is selected from IL-1 binding moieties, IL-6 binding moieties, IL-12 binding moieties, IL-23 binding moieties, or combinations thereof. In some embodiments, the compositions described herein further comprise one or more additional therapeutic agents.

[0319] dose This document provides compositions comprising one or more engineered protein constructs for the treatment (including prevention) of diseases (e.g., infectious conditions, disorders or diseases, autoimmune conditions, disorders or diseases, dermatological conditions, disorders or diseases). In some embodiments, the engineered protein construct comprises the TREM1 binding moiety described herein. In some embodiments, the engineered protein construct comprises the interleukin binding moiety described herein. In some embodiments, the engineered protein construct comprises both the TREM1 binding moiety and the interleukin binding moiety described herein. Furthermore, this document provides compositions comprising multispecific (e.g., bispecific, trispecific) antibodies or multispecific (e.g., bispecific, trispecific) molecules or antigen-binding fragments thereof for the treatment (including prevention) of diseases (e.g., infectious conditions, disorders or diseases, autoimmune conditions, disorders or diseases, dermatological 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 in treating (including preventing) infectious conditions, disorders or diseases, autoimmune conditions, disorders or diseases, dermatological conditions, disorders or diseases. In some embodiments, the composition (e.g., an antibody or an antigen-binding fragment t...

Claims

1. An engineered protein construct comprising: a first region and a second region, wherein the first region binds TREM1, a variant of TREM1, or a functional fragment of TREM1, and wherein the second region binds interleukins, wherein the interleukins comprise proteins, variants thereof, or functional fragments thereof selected from any one of the IL-1 family, IL-6 family, IL-12 family, and IL-23 family.

2. The engineered protein construct according to claim 1, wherein the engineered protein construct is an antibody, a variant of the antibody, or a functional fragment of the antibody.

3. The engineered protein construct according to any one of claims 1-2, wherein the engineered protein construct is a Fab2 antibody, a dual scFv antibody, a dual antibody, DVD-Ig, TandAb, a tandem scFv-Fc, a single-arm tandem scFv-Fc, DART, DART-Fc, or a functional fragment thereof.

4. The engineered protein construct according to any one of claims 1-3, wherein the engineered protein construct comprises a heterodimeric antibody or a functional fragment thereof.

5. The engineered protein construct according to any one of claims 1-4, wherein the engineered protein construct comprises a constant region.

6. The engineered protein construct according to any one of claims 1-5, wherein the first region comprises a TREM1-binding heavy chain variable domain.

7. The engineered protein construct according to any one of claims 1-6, wherein the first region comprises a TREM1-binding light chain variable domain.

8. The engineered protein construct according to any one of claims 1-7, wherein the second region comprises an interleukin-binding heavy chain variable domain.

9. The engineered protein construct according to any one of claims 1-8, wherein the second region comprises an interleukin-binding light chain variable domain.

10. The engineered protein construct according to any one of claims 1-9, wherein the binding affinity of the first region to TREM1 is lower than that of the second region to the interleukin.

11. The engineered protein construct of claim 10, wherein the binding affinity of the second region to the interleukin is at least twice that of the binding affinity of the first region to TREM1.

12. The engineered protein construct according to any one of claims 1-9, wherein the first region has a higher binding affinity for TREM1 than the second region has a higher binding affinity for the interleukin.

13. The engineered protein construct of claim 12, wherein the binding affinity of the first region to TREM1 is at least twice that of the binding affinity of the second region to the interleukin.

14. The engineered protein construct according to any one of claims 1-13, wherein the engineered protein construct comprises at least one of an Fc region and / or a Fab region.

15. The engineered protein construct of claim 14, wherein the Fc region comprises 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% of any one of the amino acid sequences of SEQ ID NO: 453-455.

16. The engineered protein construct according to any one of claims 1-15, wherein at least one of the first region and the second region comprises a light chain constant domain and / or a heavy chain constant domain.

17. The engineered protein construct of claim 15, wherein the heavy chain constant domain of the first region comprises an Fc region having an S354C mutation and a T366W mutation according to EU numbers, and the heavy chain constant domain of the second region comprises an Fc region having an Y349C mutation, a T366S mutation, and a Y407V mutation according to EU numbers.

18. The engineered protein construct of claim 15, wherein the heavy chain constant domain of the second region comprises an Fc region having an S354C mutation and a T366W mutation according to EU numbers, and the heavy chain constant domain of the first region comprises an Fc region having an Y349C mutation, a T366S mutation, and a Y407V mutation according to EU numbers.

19. The engineered protein construct of claim 15, wherein the Fc region comprises a constant human IgG1 heavy chain with at least one substitution selected from 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 according to EU numbers.

20. The engineered protein construct of claim 15, wherein the Fc region comprises a constant human IgG2 heavy chain with at least one substitution selected from positions C232, C233, V234, G237, P238, M252, S254, T256, H268, N297, V309, A330, and P331 according to EU numbers.

21. The engineered protein construct of claim 15, wherein the Fc region comprises a constant human IgG4 heavy chain with at least one substitution selected from positions S228, E233, F234, L235, L236, G237, S241, L248, M252, S254, T256, N297, E318, and T394 according to EU numbers.

22. The engineered protein construct according to any one of claims 1-21 exhibits pH-dependent target binding activity to a target peptide, wherein the target peptide is selected from TREM1, interleukins, their variants and functional fragments, and wherein the interleukins are selected from the IL-1 family, IL-6 family, IL-12 family and IL-23 family proteins.

23. The engineered protein construct according to any one of claims 1-22, wherein the engineered protein construct comprises an anti-inflammatory activity of 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, relative to the combined anti-inflammatory activity of the monospecific antibody binding TREM1 and the monospecific antibody binding interleukin.

24. The engineered protein construct according to any one of claims 1-23, for use in the treatment of inflammatory diseases or conditions.

25. The engineered protein construct of claim 24, wherein the inflammatory disease or condition is selected from the group consisting of: rheumatoid arthritis, juvenile arthritis, psoriatic arthritis, ankylosing spondylitis, axial spondyloarthritis, psoriasis, hidradenitis suppurativa, ulcerative colitis, Crohn's disease, necrotizing enterocolitis, sepsis, or multiple sclerosis.

26. A composition comprising an engineered protein construct according to any one of claims 1-25.

27. A composition for use in treating an inflammatory disease or condition, wherein the composition comprises: an engineered protein construct comprising a first region and a second region, wherein the first region binds TREM1, a variant thereof, or a functional fragment thereof, wherein the second region binds interleukins, wherein the interleukins comprise proteins, variants thereof, or functional fragments thereof selected from the IL-1 family, the IL-6 family, the IL-12 family, and the IL-23 family, and wherein administration of an effective amount of the composition to a subject in need results in treatment of the inflammatory disease or condition.

28. The composition for use according to claim 26, wherein the inflammatory disease or condition is selected from the group consisting of: rheumatoid arthritis, juvenile arthritis, psoriatic arthritis, ankylosing spondylitis, axial spondyloarthritis, psoriasis, hidradenitis suppurativa, ulcerative colitis, Crohn's disease, necrotizing enterocolitis, sepsis, or multiple sclerosis.

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

30. The composition for use according to claim 26, wherein the inflammatory disease or condition is psoriasis or hidradenitis suppurativa.

31. The composition for use according to claim 26, wherein the inflammatory disease or condition is ulcerative colitis, Crohn's disease, necrotizing enterocolitis, sepsis, or multiple sclerosis.

32. The composition for use according to claim 26, wherein the inflammatory disease or condition is sepsis.

33. The composition for use according to claim 26, wherein the inflammatory disease or condition is multiple sclerosis.

34. A nucleic acid encoding at least a portion of an engineered protein construct according to any one of claims 1-25.

35. A pharmaceutical composition comprising an engineered protein construct according to any one of claims 1-25 and a pharmaceutically acceptable carrier.

36. A pharmaceutical composition for use in treating an inflammatory disease or condition, wherein the pharmaceutical composition comprises: a TREM1 binding moiety, an interleukin binding moiety, and a pharmaceutically acceptable carrier, wherein the interleukin binding moiety comprises a protein selected from IL-1 binding moieties, IL-6 binding moieties, IL-12 binding moieties, and IL-23 binding moieties, and wherein administration of an effective amount of the composition to a subject in need results in treatment of the inflammatory disease or condition.

37. The pharmaceutical composition of claim 36, wherein the inflammatory disease or condition is selected from the group consisting of: rheumatoid arthritis, juvenile arthritis, psoriatic arthritis, ankylosing spondylitis, axial spondyloarthritis, psoriasis, hidradenitis suppurativa, ulcerative colitis, Crohn's disease, necrotizing enterocolitis, sepsis, or multiple sclerosis.

38. A method of treating an inflammatory disease or condition in a subject, the method comprising administering to the subject an effective amount of an engineered protein construct according to any one of claims 1-25, a composition according to any one of claims 26-33, or a pharmaceutical composition according to any one of claims 35-37, thereby treating the inflammatory disease or condition.

39. The method of claim 38, wherein the inflammatory disease or condition is associated with increased activity and / or expression of TREM1, the interleukin, one or more of its downstream inflammatory signaling proteins, or combinations thereof, relative to a subject without the inflammatory disease or condition.

40. The method of claim 38 or 39, wherein the method reduces the occurrence of Candida infection in the subject relative to a subject treated with a monospecific antibody that reduces interleukin activity.

41. A method for reducing IL-1-related inflammatory status in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition comprising an IL-1-binding moiety, a TREM1-binding moiety, and a pharmaceutically acceptable carrier, thereby reducing the subject's IL-1-related inflammatory status relative to the subject's IL-1-related inflammatory status prior to administration of the pharmaceutical composition.

42. A method for reducing IL-6-related inflammatory status in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition comprising an IL-6-binding moiety, a TREM1-binding moiety, and a pharmaceutically acceptable carrier, thereby reducing the subject's IL-6-related inflammatory status relative to the subject's IL-6-related inflammatory status prior to administration of the pharmaceutical composition.

43. A method for reducing IL-12-related inflammatory status in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition comprising an IL-12-binding moiety, a TREM1-binding moiety, and a pharmaceutically acceptable carrier, thereby reducing the subject's IL-12-related inflammatory status relative to the subject's IL-12-related inflammatory status prior to administration of the pharmaceutical composition.

44. A method for reducing IL-23-related inflammatory status in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition comprising an IL-23-binding moiety, a TREM1-binding moiety, and a pharmaceutically acceptable carrier, thereby reducing the subject's IL-23-related inflammatory status relative to the subject's IL-23-related inflammatory status prior to administration of the pharmaceutical composition.

45. A method for reducing TREM1-related inflammatory status in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition comprising a TREM1-binding portion, an interleukin-binding portion, and a pharmaceutically acceptable carrier, thereby reducing the subject's TREM1-related inflammatory status relative to the subject's TREM1-related inflammatory status prior to administration of the pharmaceutical composition.

46. ​​The method according to any one of claims 38-45, wherein the method increases the expression of at least one of the following: nicotinamide phosphoribosyltransferase (NAMPT), dehydrogenase / reductase 9 (DHRS9), cyclin-dependent kinase inhibitor 1A (CDKN1A), CD52 molecule (CD52), myotubule-associated protein 11 (MTMR11), EH domain-containing 1 (EHD1), solute carrier family 27 member 3 (SLC27A3), interleukin 24 (IL24), Pim-2 proto-oncogene serine / threonine kinase (PIM2), chitosanase 3-like 1 (CHI3L1), polypeptide N-acetylgalactosyltransferase 6 (GALNT6), acyl-CoA thioesterase 7 (ACOT7), protein containing cytokine-inducible SH2 (CISH), containing sequence-similar family 129 member A (FAM129A), polo-like kinase 3 (PLK3), containing major facilitater superfamily 12 (MFSD12), containing StAR-associated lipid transfer domain 4 (STARD4), C-type lectin domain family 12 member A (CLEC12A), CD55 molecule (Cromer blood group) (CD55) and interferon λ receptor 1 (IFNLR1).

47. The method according to any one of claims 38-46, wherein the method restores the pentose phosphate pathway (PPP).

48. The method according to any one of claims 38-47, wherein the pharmaceutical composition is selected from any one of claims 35-37.

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