WO2019112852A1 - Targeted immunotolerance - Google Patents

Targeted immunotolerance Download PDF

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Publication number
WO2019112852A1
WO2019112852A1 PCT/US2018/062780 US2018062780W WO2019112852A1 WO 2019112852 A1 WO2019112852 A1 WO 2019112852A1 US 2018062780 W US2018062780 W US 2018062780W WO 2019112852 A1 WO2019112852 A1 WO 2019112852A1
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WIPO (PCT)
Prior art keywords
polypeptide
molecule
binding
mutation
therapeutic compound
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PCT/US2018/062780
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English (en)
French (fr)
Inventor
Nathan HIGGINSON-SCOTT
Joanne L. Viney
Jyothsna Visweswaraiah
Erik Robert Sampson
Kevin Lewis Otipoby
Original Assignee
Pandion Therapeutics, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Priority claimed from PCT/US2018/034334 external-priority patent/WO2018217989A1/en
Priority claimed from US16/109,897 external-priority patent/US10174092B1/en
Priority to US17/053,979 priority Critical patent/US20210277085A1/en
Application filed by Pandion Therapeutics, Inc. filed Critical Pandion Therapeutics, Inc.
Priority to JP2020531046A priority patent/JP2021505156A/ja
Priority to EP18887096.8A priority patent/EP3720871A4/de
Publication of WO2019112852A1 publication Critical patent/WO2019112852A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/55—IL-2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70532—B7 molecules, e.g. CD80, CD86
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies

Definitions

  • the embodiments provided herein relate to, for example, methods and compositions for local or targeted immune-privilege.
  • the therapeutic compound comprises an engineered multi-specific compound, e.g., an engineered bi-specific molecule, e.g., an engineered bi-specific antibody molecule, comprising:
  • a specific targeting moiety selected from: a) a donor specific targeting moiety which, e.g., preferentially binds a donor target (preferentially as compared with binding to a recipient antigen), and is useful for providing site- specific immune privilege for a transplant tissue, e.g., an organ, from a donor; or
  • tissue specific targeting moiety which, e.g., preferentially binds a subject target tissue (preferentially as compared with subject non-target tissue), and is useful for providing site- specific immune privilege for a subject tissue undergoing unwanted immune attack, e.g., in an autoimmune disorder);
  • an immune cell inhibitory molecule binding/modulating moiety (referred to herein as an ICIM binding/modulating moiety);
  • an immunosuppressive immune cell binding/modulating moiety (referred to herein as an IIC binding/modulating moiety);
  • an effector binding/modulating moiety that, as part of a therapeutic compound, promotes an immuno-suppressive local microenvironment, e.g., by providing in the proximity of the target, a substance that inhibits or minimizes attack by the immune system of the target (referred to herein as an SM binding/modulating moiety); or
  • an immune cell stimulatory molecule binding/modulating moiety (referred to herein as an ICSM binding/modulating moiety), wherein the ICSM inhibits immune activation by, for example, blocking the interaction between a costimulatory molecule and its counterstructure.
  • An effector binding/modulating moiety can fall into more than one of classes a, b and c.
  • a CTLA4 binding molecule falls into both of categories a and b.
  • the therapeutic compound comprises an ICIM binding/modulating moiety.
  • an ICIM binding/modulating molecule and binds, and agonizes, an inhibitory molecule, e.g., an inhibitory immune checkpoint molecule, or otherwise inhibits or reduces the activity of an immune cell, e.g., a cytotoxic T cell, a B cell, NK cell, or a myeloid cell, e.g., a neutrophil or macrophage.
  • an immune cell e.g., a cytotoxic T cell, a B cell, NK cell, or a myeloid cell, e.g., a neutrophil or macrophage.
  • the therapeutic compound comprises an engineered multi-specific compound, e.g., an engineered bi-specific molecule, e.g., an engineered bi-specific antibody molecule, comprising:
  • a specific targeting moiety e.g., a donor specific targeting moiety (which binds a donor target and is useful for providing site-specific immune privilege for a transplant tissue, e.g., an organ, from a donor) or a tissue specific targeting moiety (which binds a subject tissue target and is useful for providing site-specific immune privilege for a subject tissue undergoing unwanted immune attack, e.g., in an autoimmune disorder); and
  • an effector binding/modulating moiety comprising an ICIM binding/modulating moiety that binds to an effector molecule on an immune cell, e.g., an inhibitory receptor, e.g., PD-l, wherein, upon binding of the specific targeting moiety to its target, and binding of the ICIM binding/modulating moiety to an effector molecule on the immune cell, an immune cell activity, e.g., the ability of the immune cell to mount an immune attack, is down regulated, e.g., through an inhibitory signal dependent on the clustering of effector molecules on the immune cell.
  • the engineered multi-specific compound comprises additional binding moieties so that it binds more than two specific molecules, such as, but not limited to, 3 or 4.
  • the therapeutic compound comprises an ICIM binding/modulating moiety and has one or both of the following properties: (a) the level of down regulation of an immune cell is greater when the therapeutic compound is bound to its target than when the therapeutic compound is not bound to its target; and (b) the therapeutic compound, when engaged with a cell surface inhibitory receptor, e.g., PD-l, on an immune cell, does not inhibit, or does not substantially inhibit the ability of the cell surface inhibitory receptor to bind an endogenous ligand.
  • a cell surface inhibitory receptor e.g., PD-l
  • the level of down regulation of an immune cell is greater when the therapeutic compound is bound to its target than when the therapeutic compound is not bound to its target. In embodiments, the level of down regulation by target bound therapeutic compound is equal to or greater than 1.5-fold, 2-fold, 4-fold, 8-fold or lO-fold greater than what is seen when it is not bound to its target. In embodiments, therapeutic compound does not, or does not significantly down regulate immune cells when it is not bound to target. Thus, indiscriminant or unwanted agonism of an inhibitory receptor, e.g., PD-l, is minimized or eliminated.
  • an inhibitory receptor e.g., PD-l
  • the therapeutic compound when engaged with a cell surface inhibitory receptor, e.g., PD-l, on an immune cell, does not inhibit, or does not substantially inhibit the ability of the cell surface inhibitory receptor to bind an endogenous ligand.
  • a cell surface inhibitory receptor e.g., PD-l
  • the therapeutic compound can bind to the PD-L 1/2 binding site on PD-l.
  • indiscriminant or unwanted antagonism of an inhibitory receptor, e.g., PD-l is minimized or eliminated.
  • binding of the therapeutic compound to an inhibitory receptor, e.g. PD-l, on an immune cell does not impede, or substantially impede, the ability of the inhibitory receptor to bind a natural ligand, e.g., PD-L1.
  • binding of the therapeutic compound to an inhibitory receptor, e.g. PD-l, on an immune reduces binding of a natural ligand, e.g., PD-L1, by less than 50, 40, 30, 20, 10, or 5% of what is seen in the absence of therapeutic compound.
  • the therapeutic compound comprises an ICIM binding/modulating moiety and, when administered to a subject at a therapeutically effective dose, does not result in unacceptable levels of systemic immune suppression, as would be possible if indiscriminant agonism of the inhibitory receptor in all immune cells of a type, e.g., all T cells, occurred, or unacceptable levels of systemic immune activation, as would be possible if the therapeutic compound antagonized the interaction of the inhibitory receptor with its natural ligand.
  • a therapeutic compound comprising an ICIM binding/modulating moiety can exist in any one of four states: i) unbound and in free solution; ii) bound to only an inhibitory receptor expressed on the surface of an immune cell, e.g., a T cell, through the ICIM binding/modulating moiety; iii) bound to only the surface of the target transplant or subject tissue through the targeting moiety; and iv) bound to both the surface of target transplant or subject tissue through the targeting moiety and to an inhibitory receptor expressed by an immune cell, e.g., a T cell, through the ICIM binding/modulating moiety.
  • the therapeutic compound When the therapeutic compound is bound only to the target transplant or subject tissue (iii) through the targeting moiety, it has no, or no substantial, effect on the target transplant or tissue.
  • the therapeutic compound When the therapeutic compound is bound to the target transplant or tissue through the targeting moiety and bound to an inhibitory receptor expressed by an immune cell, e.g., a T cell, through the ICIM binding/modulating moiety (iv), it creates immune privilege at the target organ or tissue. While not wishing to be bound by theory, is believed that this is achieved by the target transplant or donor tissue multimerizing the therapeutic compound molecules on its surface, e.g., by immobilizing a plurality of therapeutic compound molecules at a high density and valency.
  • the multimerization of the therapeutic compound molecules allows the ICIM binding/modulating moieties of the therapeutic compounds to promote clustering of inhibitory receptors expressed on the surface of the immune cell, e.g., a pathogenic T cell, and transmission of an inhibitory signal functioning to silence or down-regulate the immune cell.
  • a therapeutic compound comprising an ICIM binding/modulating moiety comprising a PD-L1 molecule, or an anti-PD-l Ab, can be used.
  • Binding of a plurality of the therapeutic compound molecules to the target results in multimerization of the therapeutic compound molecules, which in turn, by virtue of the PD-L1 molecule, or a functional anti-PD-l antibody molecule, leads to clustering of PD-l on the T cell. If that clustering occurs in the context of antigen presentation by the target MHC, to T cell receptor on the T cell, a negative signal is generated and the T cell will be inactivated.
  • the ICIM binding/modulating moiety e.g., a functional antibody molecule, binds the effector molecule but does not inhibit, or substantially inhibit, interaction of the effector molecule with its native ligand(s).
  • the therapeutic compound comprises an IIC binding/modulating moiety, which binds and recruits an immune suppressive immune cell, e.g., a Treg, e.g., a Foxp3+CD25+ Treg, to the proximity of the target tissue.
  • an immune suppressive immune cell e.g., a Treg, e.g., a Foxp3+CD25+ Treg
  • the therapeutic compound comprises a SM binding/modulating moiety, which modulates, e.g., binds and inhibits, sequesters, degrades or otherwise neutralizes a substance, e.g., a soluble molecule that modulates an immune response, e.g., ATP or AMP.
  • a SM binding/modulating moiety which modulates, e.g., binds and inhibits, sequesters, degrades or otherwise neutralizes a substance, e.g., a soluble molecule that modulates an immune response, e.g., ATP or AMP.
  • the therapeutic compound comprises a targeting moiety that is specific for a target on an immune cell.
  • the target is as described herein.
  • the target is MAdCAM.
  • the targeting moiety is an antibody that binds to MAdCAM.
  • the therapeutic compound comprises an ICSM binding/modulating moiety, which binds a stimulatory molecule, e.g., a costimulatory molecule.
  • a stimulatory molecule e.g., a costimulatory molecule.
  • the ICSM inhibits the costimulatory molecule counterstructure by.
  • Binding/modulating either the costimulatory molecule or the costimulatory molecule counterstructure can serve to down regulate the ability of an immune cell to mount an immune response.
  • the ICSM binding/modulating moiety can bind a stimulatory, e.g., costimulatory molecule on an immune cell, e.g., 0X40 on T cells , or the counter member of the stimulatory molecule e.g. OX40L on another cell, such as, but not limited to, immune cells such as NK cells, mast cells, dendritic cells, or, for example, non-immune cells such as endothelial cells, or smooth muscle cells.
  • the therapeutic compound comprises a donor specific targeting moiety and provides site-specific immune privilege for donor transplant tissue implanted in a subject.
  • the therapeutic compound comprises a tissue specific targeting moiety and provides site-specific immune privilege for a tissue of a subject, e.g., a tissue afflicted with an unwanted immune response in an autoimmune disorder.
  • the targeting moiety is specific for the donor transplant or subject tissue to be protected from the immune system.
  • the effector molecule binding moiety comprises a de novo generated binding domain, e.g. a functional antibody molecule.
  • the effector binding/modulating moiety comprises amino acid sequence deriving from the natural ligand that recognizes an inhibitory receptor expressed on the surface of an immune cell, e.g., a T cell.
  • the therapeutic compound silences immune cells, e.g., T cells, proximal to the transplant or donor tissue to be protected but does not silence immune cells, e.g., T cells, not proximal to the target, as the therapeutic compound requires the presence of the target transplant or donor tissue for function.
  • immune cells e.g., T cells
  • T cells proximal to the transplant or donor tissue to be protected
  • the therapeutic compound binds only to the inhibitory receptor expressed by the immune cell, e.g., T cell, in which case there is no functional consequence.
  • Methods and therapeutic compounds described here are based at least in part on providing site-specific immune-privilege.
  • Therapeutic compounds and method of using them described herein allow the minimization, e.g., the reduction or elimination of, non-site specific systemic administration of immune-suppressive therapeutic agents in clinical settings, e.g., where reversal and suppression of an immune response is desired, such as in autoimmune diseases or tissue, e.g., organ, transplant.
  • autoimmune diseases or tissue e.g., organ, transplant.
  • broadly acting immunosuppressants have the undesirable effect of reducing the patient’s systemic immune system function.
  • a therapeutic compound is provided as provided herein.
  • the compound comprises a i) a specific targeting moiety selected from: a) a donor specific targeting moiety which, e.g., preferentially binds a donor target; or b) a tissue specific targeting moiety which, e.g., preferentially binds target tissue of a subject; and ii) an effector binding/modulating moiety selected from: (a) an immune cell inhibitory molecule
  • IC binding/modulating moiety an immunosuppressive immune cell binding/modulating moiety
  • effector binding/modulating moiety that, as part of a therapeutic compound, promotes an immuno- suppressive local microenvironment, e.g., by providing in the proximity of the target, a substance that inhibits or minimizes attack by the immune system of the target (SM binding/modulating moiety).
  • the effector binding/modulating moiety comprises an ICIM binding/modulating moiety. In some embodiments, the effector binding/modulating moiety comprises an ICIM binding/modulating moiety comprising an inhibitory immune checkpoint molecule ligand molecule. In some embodiments, the inhibitory immune molecule counter ligand molecule comprises a PD-L1 molecule. In some embodiments, the ICIM is wherein the inhibitory immune molecule counter ligand molecule engages a cognate inhibitory immune checkpoint molecule selected from PD-l, KIR2DL4, LILRB1, LILRB, or CTLA-4. In some embodiments, the ICIM is an antibody.
  • the ICIM comprises an antibody that binds to PD-l, KIR2DL4, LILRB 1, LILRB, or CTLA-4.
  • the ICIM binding/modulating moiety which comprises a functional antibody molecule to a cell surface inhibitory molecule.
  • the cell surface inhibitory molecule is an inhibitory immune checkpoint molecule.
  • the inhibitory immune checkpoint molecule is selected from PD-l, KIR2DL4, LILRB1, LILRB2, CTLA-4, or selected from Table 1.
  • the effector binding/modulating moiety comprises an IIC binding/modulating moiety.
  • the compound has the formula from N-terminus to C-terminus: Rl— Linker Region A— R2 or R3— Linker Region B— R4,
  • Rl, R2, R3, and R4 each independently comprises an effector binding/modulating moiety, e.g., an ICIM binding/modulating moiety, an IIC binding/modulating moiety, ICSM binding/modulating moiety, or an SM binding/modulating moiety; a specific targeting moiety; or is absent; provided that an effector binding/modulating moiety and a specific targeting moiety are present.
  • an effector binding/modulating moiety e.g., an ICIM binding/modulating moiety, an IIC binding/modulating moiety, ICSM binding/modulating moiety, or an SM binding/modulating moiety
  • a specific targeting moiety e.g., a specific targeting moiety, or is absent; provided that an effector binding/modulating moiety and a specific targeting moiety are present.
  • polypeptides comprising a targeting moiety that binds to a target cell and an effector binding/modulating moiety, wherein the effector binding/modulating moiety is a IL-2 mutein polypeptide (IL-2 mutein), which is a mutant IL-2 protein, are provided.
  • the targeting moiety comprises an antibody that binds to a target protein on the surface of a target cell.
  • the polypeptide comprises two polypeptide chains as provided for herein.
  • the first chain comprises a VH domain and the second chain comprises a VL domain of an antibody that binds to the target cell or a protein that is expressed on the target cell, such as, but not limited to, MAdCAM.
  • the targeting moiety is an antibody that binds to MAdCAM.
  • the targeting moiety binds to OAT1 (SLC22A6) and OCT2 (SLC22A2).
  • the targeting moiety is an antibody that binds to OAT1 (SLC22A6) and OCT2 (SLC22A2).
  • the targeting moiety does not bind to OAT1 (SLC22A6) and OCT2 (SLC22A2).
  • the OCT2 referenced herein is not the transcription factor, but rather is the surface protein expressed in kidney tissue.
  • the targeting moiety is a moiety that specifically binds to a protein found in the pancreas.
  • the targeting moiety binds to FXYD2, TSPAN7, DPP6, HEPACAM2, TMEM27, or GPR119. In some embodiments, the targetin moiety does not bind to FXYD2, TSPAN7, DPP6, HEPACAM2, TMEM27, or GPR119. In some embodiments, the targeting moiety is antibody that binds to FXYD2, TSPAN7, DPP6, HEPACAM2, TMEM27, or GPR119.
  • the polypeptide comprises a first chain and a second chain that form the polypeptide or therapeutic compound, wherein
  • the first chain comprises:
  • V H -H c -Linker-Ci wherein V H is a variable heavy domain that binds to the target cell with a V L domain of the second chain; H c is a heavy chain of antibody comprising CH1-CH2-CH3 domain, the Linker is a glycine/serine amino acid sequence as provided herein or is absent, and Ci is a IL-2 mutein that can be fused to a Fc protein in either the N-terminal or C-terminal orientation as provided for herein, wherein there can be a glycine/serine linker linking the IL-2 mutein to the Fc protein; and
  • the second chain comprises:
  • V L is a variable light chain domain that binds to the target cell with the V H domain of the first chain
  • the Lc domain is a light chain CK domain.
  • the first chain comprises Ci-Linker-V H -H c , with the variables as defined above.
  • the polypeptide comprises the formula of Ci-linker-CH2-CH3- Linker-scFv, wheren Ci and the Linker are as defined above and herein, the CH2 and CH3 are heavy chain domains and the scFv is a single chain antibody like fragment that acts as the targeting moiety to bind to tissue targets as provided for herein.
  • the mutein is fused to the Fc region as provided herein and one or more of the linkers are absent.
  • the Linker is a gly cine/ serine linker as provided for herein.
  • the linker is a peptide sequence.
  • methods of treating auto-immune diseases or conditions comprising administering one or more of the therapeutic compounds or polypeptides provided herein.
  • methods of treating diseases or conditions described herein are provided herein, the methods comprising administering one or more of the therapeutic compounds or polypeptides provided herein.
  • methods of treating a subject with inflammatory bowel disease comprising administering a therapeutic compound or polypeptides provided herein to the subject to treat the inflammatory bowel disease.
  • the subject has Crohn’s disease and or ulcerative colitis.
  • methods of treating a subject with auto-immune hepatitis comprising administering a therapeutic compound or polypeptides as provided herein to the subject to treat the auto-immune hepatitis.
  • methods of treating primary sclerosing cholangitis comprising administering a therapeutic compound or polypeptides as provided herein to the subject to treat the primary sclerosing cholangitis.
  • methods of treating e.g ., reducing) inflammation in the intestine are provided, the methods comprising administering a therapeutic compound or polypeptides as provided herein to the subject to treat the inflammation in the intestine.
  • the inflammation is in the small intestine.
  • the inflammation is in the large intesting.
  • the inflammation is in the bowel or colon.
  • methods of treating e.g., reducing inflammation in the pancreas are provided, the methods comprising administering a therapeutic compound or polypeptides as provided herein to the subject to treat the inflammation in the pancreas.
  • the methods treat pancreatitis.
  • methods of treating Type 1 diabetes comprising administering a therapeutic compound or polypeptides as provided herein to the subject to treat the Type 1 diabetes.
  • methods of treating a transplant subject comprising administering a therapeutically effective amount of a therapeutic compound or polypeptides as provided herein to the subject, thereby treating a transplant (recipient) subject.
  • methods of treating GVHD in a subject having a transplanted a donor tissue comprising administering a therapeutically effective amount of a therapeutic compound or polypeptides as provided herein to the subject.
  • methods of treating a subject having, or at risk, or elevated risk, for having, an autoimmune disorder comprising administering a therapeutically effective amount of a therapeutic compound or polypeptides as provided herein, thereby treating the subject.
  • FIG. 1 depicts non-limiting embodiments of the therapeutic compounds provided herein.
  • FIG. 2 depicts a non-limiting illustration of how a therapeutic compound provided herein could function.
  • FIG. 3 depicts a non-limiting illustration of the therapeutic compounds provided herein.
  • FIG. 3 A depicts a non-limiting illustration of the therapeutic compounds provided herein.
  • FIG. 4 depicts a non-limiting illustration of the therapeutic compounds provided herein.
  • FIG. 5 depicts a non-limiting illustration of the therapeutic compounds provided herein.
  • FIG. 6 depicts a non-limiting illustration of the therapeutic compounds provided herein.
  • FIG. 7 depicts a non-limiting illustration of the therapeutic compounds provided herein.
  • FIG. 8 depicts a non-limiting illustration of the therapeutic compounds provided herein.
  • FIG. 9 depicts a non-limiting illustration of the therapeutic compounds provided herein.
  • FIG. 10 depicts a non-limiting illustration of the therapeutic compounds provided herein.
  • FIG. 11 depicts a non-limiting illustration of the therapeutic compounds provided herein.
  • FIG. 12 depicts a non-limiting illustration of the therapeutic compounds provided herein.
  • FIG. 13 depicts a non-limiting illustration of the therapeutic compounds provided herein.
  • FIG. 14 depicts a non-limiting illustration of the therapeutic compounds provided herein.
  • FIG. 15 depicts a non-limiting illustration of the therapeutic compounds provided herein.
  • FIG. 16 depicts a non-limiting illustration of the therapeutic compounds provided herein.
  • FIG. 17 depicts a non-limiting illustration of the therapeutic compounds provided herein.
  • FIG. 18 depicts a non-limiting illustration of the therapeutic compounds provided herein.
  • FIG. 19 depicts a non-limiting illustration of the therapeutic compounds provided herein.
  • the term“about” means that the numerical value is approximate and small variations would not significantly affect the practice of the disclosed embodiments. Where a numerical limitation is used, unless indicated otherwise by the context,“about” means the numerical value can vary by ⁇ 10% and remain within the scope of the disclosed embodiments.
  • animal includes, but is not limited to, humans and non-human vertebrates such as wild, domestic, and farm animals.
  • “contacting” means bringing together of two elements in an in vitro system or an in vivo system.
  • “contacting” a therapeutic compound with an individual or patient or cell includes the administration of the compound to an individual or patient, such as a human, as well as, for example, introducing a compound into a sample containing a cellular or purified preparation containing target.
  • the terms“comprising” (and any form of comprising, such as“comprise”, “comprises”, and“comprised”),“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, unrecited elements or method steps. Any composition or method that recites the term“comprising” should also be understood to also describe such compositions as consisting, consisting of, or consisting essentially of the recited components or elements.
  • the term“fused” or“linked” when used in reference to a protein having different domains or heterologous sequences means that the protein domains are part of the same peptide chain that are connected to one another with either peptide bonds or other covalent bonding.
  • the domains or section can be linked or fused directly to one another or another domain or peptide sequence can be between the two domains or sequences and such sequences would still be considered to be fused or linked to one another.
  • the various domains or proteins provided for herein are linked or fused diretctly to one another or a linker sequences, such as the glycine/serine sequences described herein link the two domains together.
  • the term“individual,”“subject,” or“patient,” used interchangeably, means any animal, including mammals, such as mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, such as humans.
  • the term“inhibit” refers to a result, symptom, or activity being reduced as compared to the activity or result in the absence of the compound that is inhibiting the result, symptom, or activity.
  • the result, symptom, or activity is inhibited by about, or, at least, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%.
  • An result, symptom, or activity can also be inhibited if it is completely elimination or extinguished.
  • the phrase“in need thereof’ means that the subject has been identified as having a need for the particular method or treatment. In some embodiments, the identification can be by any means of diagnosis. In any of the methods and treatments described herein, the subject can be in need thereof. In some embodiments, the subject is in an environment or will be traveling to an environment in which a particular disease, disorder, or condition is prevalent.
  • the phrase“integer from X to Y” means any integer that includes the endpoints.
  • the phrase“integer from X to Y” means 1, 2, 3, 4, or 5.
  • the term“mammal” means a rodent (i.e., a mouse, a rat, or a guinea pig), a monkey, a cat, a dog, a cow, a horse, a pig, or a human. In some embodiments, the mammal is a human.
  • therapeutic compounds are provided herein. In some embodiments, therapeutic compounds are provided herein.
  • the therapeutic compound is a protein or a polypeptide, that has multiple chains that interact with one another.
  • the polypeptides can interact with one another through non- covalent interactions or covalent interactions, such as through disulfide bonds or other covalent bonds. Therefore, if an embodiment refers to a therapeutic compound it can also be said to refer to a protein or polypeptide as provided for herein and vice versa as the context dictates.
  • the phrase“ophthalmically acceptable” means having no persistent detrimental effect on the treated eye or the functioning thereof, or on the general health of the subject being treated.
  • transient effects such as minor irritation or a“stinging” sensation are common with topical ophthalmic administration of drugs and the existence of such transient effects is not inconsistent with the composition, formulation, or ingredient (e.g., excipient) in question being“ophthalmically acceptable” as herein defined.
  • the pharmaceutical compositions can be ophthalmically acceptable or suitable for ophthalmic administration.
  • Specific binding or “specifically binds to” or is “specific for” a particular antigen, target, or an epitope means binding that is measurably different from a non-specific interaction. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which generally is a molecule of similar structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target.
  • Specific binding for a particular antigen, target, or an epitope can be exhibited, for example, by an antibody having a K D for an antigen or epitope of at least about 10 4M , at least about 10 5M , at least about 10 6 M , at least about 10 7M , at least about 10 8M , at least about 10 9M , alternatively at least about 10 10 M , at least about 10 11M , at least about 10 12M , or greater, where K D refers to a dissociation rate of a particular antibody-target interaction.
  • an antibody that specifically binds an antigen or target will have a K D that is, or at least, 2-, 4-, 5-, 10-, 20-, 50-, 100-, 500-, 1000-, 5,000-, 10,000-, or more times greater for a control molecule relative to the antigen or epitope.
  • specific binding for a particular antigen, target, or an epitope can be exhibited, for example, by an antibody having a KA or K a for a target, antigen, or epitope of at least 2-, 4-, 5-, 20-, 50-, 100-, 500-, 1000-, 5,000-, 10,000- or more times greater for the target, antigen, or epitope relative to a control, where K A or K a refers to an association rate of a particular antibody-antigen interaction.
  • the therapeutic compounds and compositions can be used in methods of treatment as provided herein.
  • the terms“treat,”“treated,” or“treating” mean both therapeutic treatment and prophylactic measures wherein the object is to slow down (lessen) an undesired physiological condition, disorder or disease, or obtain beneficial or desired clinical results.
  • beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; diminishment of extent of condition, disorder or disease; stabilized (i.e., not worsening) state of condition, disorder or disease; delay in onset or slowing of condition, disorder or disease progression; amelioration of the condition, disorder or disease state or remission (whether partial or total), whether detectable or undetectable; an amelioration of at least one measurable physical parameter, not necessarily discernible by the patient; or enhancement or improvement of condition, disorder or disease.
  • Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment.
  • therapeutic compounds e.g., therapeutic protein molecules, e.g., fusion proteins, including a targeting moiety and an effector binding/modulating moiety, typically as separate domains. Also provided are methods of using and making the therapeutic compounds.
  • the targeting moiety serves to localize the therapeutic compound, and thus the effector binding/modulating moiety, to a site at which immune-privilege is desired.
  • the effector binding/modulating moiety comprises one or more of: (a) an immune cell inhibitory molecule binding/modulating moiety (an ICIM binding/modulating moiety): (b) an immunosuppressive immune cell binding/modulating moiety (an IIC binding/modulating moiety); (c) a soluble molecule binding/modulating moiety (a SM binding/modulating moiety) or (d) a molecule that blocks or inhibits immune cell stimulatory molecule binding/modulating moiety (referred to herein as an ICSM binding/modulating moiety).
  • the ICSM inhibits immune activation by, for example, blocking the interaction between a costimulatory molecule and its counterstructure.
  • a therapeutic compound comprises: (a) and (b); (a) and (c); (a) and (d); (b) and (c); (b) and (d); (c) and (d); or (a), (b), (c), and (d).
  • the present disclosure provides, for example, molecules that can act as PD-l agonists. Without being bound to any particular theory, agonism of PD-l inhibits T cell
  • activation/signaling can be accomplished by different mechanisms.
  • cross- linking can lead to agonism, bead-bound, functional PD-l agonists have been described
  • Crosslinking of PD-l with two mAbs that bind non-overlapping epitopes induces PD-l signaling (Davis, US 2011/0171220), which is hereby incorporated by reference.
  • PD-l signaling Daavis, US 2011/0171220
  • Another example is illustrated through the use of a goat anti-PD-l antiserum (e.g. AF1086, R&D Systems) which is hereby incorporated by reference, which acts as an agonist when soluble (Said et ak, 2010, Nat Med) which is hereby incorporated by reference.
  • Non-limiting examples of PD-l agonists that can be used in the present embodiments include, but are not limited to, UCB clone 19 or clone 10, PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4 and PD1AB-5, PD1AB-6 (Anaptys/Celgene), PD1-17, PD 1-28, PD 1-33 and PD 1-35 (Collins et al, US 2008/0311117 Al
  • the PD-l agonist antibodies can be antibodies that block binding of PD-L 1 to PD-l.
  • the PD-l agonist antibodies can be antibodies that do not block binding of PD-L1 to PD-l.
  • PD-l agonism can be measured by any method, such as the methods described in the examples.
  • cells can be constructed that express, including stably express, constructs that include a human PD-l polypeptide fused to a b-galactosidase“Enzyme donor” and 2) a SHP-2 polypeptide fused to a b-galactosidase“Enzyme acceptor.”
  • SHP-2 is recruited to PD-l.
  • the enzyme acceptor and enzyme donor form a fully active b-galactosidase enzyme that can be assayed.
  • the assay does not directly show PD-l agonism, but shows activation of PD-l signaling.
  • PD-l agonism can also be measured by measuring inhibition of T cell activation because, without being bound to any theory, PD-l agonism inhibits anti-CD3 -induced T cell activation.
  • PD-l agonism can be measured by preactivating T cells with PHA (for human T cells) or ConA (for mouse T cells) so that they express PD-l .
  • the cells can then be reactivated with anti-CD3 in the presence of anti-PD-l (or PD-L1) for the PD-l agonism assay.
  • T cells that receive a PD-l agonist signal in the presence of anti-CD3 will show decreased activation, relative to anti-CD3 stimulation alone.
  • Activation can be readout by proliferation or cytokine production (IL-2, IFNg, IL-17) or other markers, such as CD69 activation marker.
  • PD-l agonism can be measured by either cytokine production or cell proliferation. Other methods can also be used to measure PD-l agonism.
  • PD-l is Ig superfamily member expressed on activated T cells and other immune cells.
  • the natural ligands for PD-l appear to be PD-L1 and PD-L2.
  • an inhibitory signaling cascade is initiated, resulting in attenuation of the activated T effector cell function.
  • checkpoint inhibition blocking the interaction between PD-l on a T cell, and PD-L1/2 on another cell (eg tumor cell) with a PD-l antagonist is known as checkpoint inhibition, and releases the T cells from inhibition.
  • PD-l agonist antibodies can bind to PD-l and send an inhibitory signal and attenuate the function of a T cell.
  • PD-l agonist antibodies can be incorporated into various embodiments described herein as an effector molecule binding/modulating moiety, which can accomplish localized tissue-specific immunomodulation when paired with a targeting moiety.
  • the effector molecule binding/modulating moiety can provide an immunosuppressive signal or environment in a variety of ways.
  • binding/modulating moiety comprises an ICIM binding/modulating moiety that directly binds and (under the appropriate conditions as described herein) activates an inhibitory receptor expressed by immune cells responsible for driving disease pathology.
  • the effector binding/modulating moiety comprises and IIC binding/modulating moiety and binds and accumulates immunosuppressive immune cells.
  • the accumulated immune suppressive cells promote immune privilege.
  • the effector binding/modulating moiety comprises an SM binding/modulating moiety which manipulates the surrounding microenvironment to make it less permissible for the function of immune cells, e.g., immune cells driving disease pathology.
  • the SM binding/modulating moiety depletes an entity that promotes immune attack or activation.
  • the effector binding/modulating moiety comprises an ICSM binding/modulating moiety that binds a member of a pair of stimulatory molecules, e.g., costimulatory molecules, and inhibits the interaction between the costimulatory molecule and the costimulatory molecule counterstructure, such as, but not limited to, 0X40 or CD30 or CD40 and OX40L, or CD30L or CD40L and inhibits the immune stimulation of a cell, such as, but not limited to, a T cell, B cell, NK cell, or other immune cell comprising a member of the pair.
  • a pair of stimulatory molecules e.g., costimulatory molecules
  • the costimulatory molecule counterstructure such as, but not limited to, 0X40 or CD30 or CD40 and OX40L, or CD30L or CD40L
  • the immune stimulation of a cell such as, but not limited to, a T cell, B cell, NK cell, or other immune cell comprising a member of the pair.
  • the targeting moiety and effector binding/modulating moiety are physically tethered, covalently or non-covalently, directly or through a linker entity, to one another, e.g., as a member of the same protein molecule in a therapeutic protein molecule.
  • the targeting and effector moieties are provided in a therapeutic protein molecule, e.g., a fusion protein, typically as separate domains.
  • the targeting moiety, the effector binding/modulating moiety, or both each comprises a single domain antibody molecule, e.g., a camelid antibody VHH molecule or human soluble VH domain . It may also contain a single- chain fragment variable (scFv) or a Fab domain.
  • the therapeutic protein molecule, or a nucleic acid, e.g., an mRNA or DNA, encoding the therapeutic protein molecule can be administered to a subject.
  • the targeting and effector molecule binding/modulating moieties are linked to a third entity, e.g., a carrier, e.g., a polymeric carrier, a dendrimer, or a particle, e.g., a nanoparticle.
  • the therapeutic compounds can be used to down regulate an immune response at or in a tissue at a selected target or site while having no or substantially less immunosuppressive function systemically.
  • the target or site can comprise donor tissue or autologous tissue.
  • an allograft tissue e.g., a tissue described herein, e.g., an allograft liver, an allograft kidney, an allograft heart, an allograft pancreas, an allograft thymus or thymic tissue, allograft skin, or an allograft lung, with therapeutic compounds disclosed herein.
  • the treatment minimizes rejection of, minimizes immune effector cell mediated damage to, prolongs acceptance of, or prolongs the functional life of, donor transplant tissue.
  • GVHD graft versus host disease
  • the method provides tolerance to, minimization of the rejection of, minimization of immune effector cell mediated damage to, or prolonging a function of, subject tissue.
  • the therapeutic compound includes a targeting moiety that targets, e.g., specifically targets, the tissue under, or at risk for, autoimmune attack.
  • Non-limiting exemplary tissues include, but are not limited to, the pancreas, myelin, salivary glands, synoviocytes, and myocytes.
  • beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; diminishment of extent of condition, disorder or disease; stabilized (i.e., not worsening) state of condition, disorder or disease; delay in onset or slowing of condition, disorder or disease progression;
  • Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment.
  • “treatment of an auto-immune disease/disorder” means an activity that alleviates or ameliorates any of the primary phenomena or secondary symptoms associated with the auto-immune disease/disorder or other condition described herein.
  • the various disease or conditions are provided herein.
  • the therapeutic treatment can also be administered prophylactically to preventing or reduce the disease or condition before the onset.
  • administration of the therapeutic compound begins after the disorder is apparent. In some embodiments, administration of the therapeutic compound, begins prior to onset, or full onset, of the disorder. In some embodiments, administration of the therapeutic compound, begins prior to onset, or full onset, of the disorder, e.g., in a subject having the disorder, a high-risk subject, a subject having a biomarker for risk or presence of the disorder, a subject having a family history of the disorder, or other indicator of risk of, or asymptomatic presence of, the disorder. For example, In some embodiments, a subject having islet cell damage but which is not yet diabetic, is treated.
  • the targeting moiety functions to bind and accumulate the therapeutic to a target selectively expressed at the anatomical site where immune privilege is desired.
  • the target moiety binds to a target, e.g., an allelic product, present in the donor tissue but not the recipient.
  • the targeting moiety binds a target preferentially expressed at the anatomical site where immune privilege is desired, e.g., in the pancreas.
  • the targeting moiety targets the host tissue, and protects the host against attack from transplanted immune effector cells derived from transplanted tissue.
  • the effector binding/modulating moiety serves to deliver an immunosuppressive signal or otherwise create an immune privileged environment.
  • Antibody molecule refers to a polypeptide, e.g., an immunoglobulin chain or fragment thereof, comprising at least one functional immunoglobulin variable domain sequence.
  • An antibody molecule encompasses antibodies (e.g., full-length antibodies) and antibody fragments.
  • an antibody molecule comprises an antigen binding or functional fragment of a full length antibody, or a full length immunoglobulin chain.
  • a full-length antibody is an immunoglobulin (Ig) molecule (e.g., an IgG antibody) that is naturally occurring or formed by normal immunoglobulin gene fragment recombinatorial processes).
  • an antibody molecule refers to an immunologically active, antigen-binding portion of an immunoglobulin molecule, such as an antibody fragment.
  • An antibody fragment e.g., functional fragment, comprises a portion of an antibody, e.g., Fab, Fab', F(ab')2, F(ab)2, variable fragment (Fv), domain antibody (dAb), or single chain variable fragment (scFv).
  • a functional antibody fragment binds to the same antigen as that recognized by the intact (e.g., full-length) antibody.
  • antibody fragment or“functional fragment” also include isolated fragments consisting of the variable regions, such as the“Fv” fragments consisting of the variable regions of the heavy and light chains or recombinant single chain polypeptide molecules in which light and heavy variable regions are connected by a peptide linker (“scFv proteins”).
  • an antibody fragment does not include portions of antibodies without antigen binding activity, such as Fc fragments or single amino acid residues.
  • Exemplary antibody molecules include full length antibodies and antibody fragments, e.g., dAb (domain antibody), single chain, Fab, Fab’, and F(ab’)2 fragments, and single chain variable fragments (scFvs).
  • antibody molecule also encompasses whole or antigen binding fragments of domain, or single domain, antibodies, which can also be referred to as“sdAb” or“VHH.”
  • Domain antibodies comprise either V H or V L that can act as stand-alone, antibody fragments. Additionally, domain antibodies include heavy-chain-only antibodies (HCAbs).
  • Domain antibodies also include a CH2 domain of an IgG as the base scaffold into which CDR loops are grafted. It can also be generally defined as a polypeptide or protein comprising an amino acid sequence that is comprised of four framework regions interrupted by three complementarity determining regions. This is represented as FR1- CDR1 -FR2-CDR2-FR3-CDR3-FR4.
  • sdAbs can be produced in camelids such as llamas, but can also be synthetically generated using techniques that are well known in the art.
  • the numbering of the amino acid residues of a sdAb or polypeptide is according to the general numbering for VH domains given by Rabat et al.
  • FR1 of a sdAb comprises the amino acid residues at positions 1-30
  • CDR1 of a sdAb comprises the amino acid residues at positions 31-36
  • FR2 of a sdAb comprises the amino acids at positions 36-49
  • CDR2 of a sdAb comprises the amino acid residues at positions 50-65
  • FR3 of a sdAb comprises the amino acid residues at positions 66- 94
  • CDR3 of a sdAb comprises the amino acid residues at positions 95-102
  • FR4 of a sdAb comprises the amino acid residues at positions 103-113.
  • Domain antibodies are also described in W02004041862 and
  • the domain antibodies can be a targeting moiety as described herein.
  • Antibody molecules can be monospecific (e.g., monovalent or bivalent), bispecific (e.g., bivalent, trivalent, tetravalent, pentavalent, or hexavalent), trispecific (e.g., trivalent, tetravalent, pentavalent, hexavalent), or with higher orders of specificity (e.g, tetraspecific) and/or higher orders of valency beyond hexavalency.
  • An antibody molecule can comprise a functional fragment of a light chain variable region and a functional fragment of a heavy chain variable region, or heavy and light chains may be fused together into a single polypeptide. Examples of formats for multispecific therapeutic compounds, e.g., bispecific antibody molecules are shown in the following non-limiting examples. Although illustrated with antibody molecules, they can be used as platforms for therapeutic molecules that include other non antibody moieties as specific binding or effector moieties. In some embodiments, these non limiting examples are based upon either a symmetrical or asymmetrical Fc formats
  • the figures illustrate non-limiting and varied symmetric homodimer approach.
  • the dimerization interface centers around human IgGl CH2- CH3 domains, which dimerize via a contact interface spanning both CH2/CH2 and CH3/CH3.
  • the resulting bispecific antibodies shown have a total valence comprised of four binding units with two identical binding units at the N-terminus on each side of the dimer and two identical units at the C-terminus on each side of the dimer. In each case the binding units at the N- terminus of the homo-dimer are different from those at the C-terminus of the homo-dimer.
  • bivalency for both an inhibitory T cell receptor at either terminus of the molecule and bivalency for a tissue tethering antigen can be achieved at either end of the molecule.
  • the N-terminus of the homodimer contains two identical Fab domains comprised of two identical light chains, which are separate polypeptides, interfaced with the n-terminal VH-CH1 domains of each heavy chain via the VH/VL interaction and Ckappa or Clambda interaction with CH1.
  • the native disulphide bond between the Ckappa or Clambda with CH1 is present providing a covalent anchor between the light and heavy chains.
  • scFvs may be configured to be from N to C terminus either VH-Linker-VL or VL-Linker-VH.
  • a non-limiting example of a molecule that has different binding regions on the different ends is where, one end is a PD-l agonist and the antibody that provides target specificity is an anti-MAdCAM-l antibody. This can be illustrated as shown, for example, in FIG. 3 A, which illustrates the molecules in different orientations.
  • the MAdCAM antibody is a blocking or non-blocking antibody as described elsewhere herein.
  • MAdCAM has been shown to interact with the headpiece of the integrin a4b7 expressed on lymphocytes via multiple residues within its two Ig superfamily I-set domains and the atomic level structural basis for that interaction has been described (Viney JL et al. (1996). J Immunol. 157, 2488-2497; Yu Y et al (2013). J Biol Chem. 288, 6284-6294; Yu Y et al (2012). J Cell Biol. 196, 131-146, each of which is incorporated by reference in its entirety).
  • the MAdCAM/a4b7 interaction is shown to be of a lower functional affinity and permits rolling adhesion of lymphocytes, whereas in low Ca2+ but higher Mg2+ or Mn2+ which activates the integrin, the MAdCAM/c ⁇ 7 interaction is of a higher functional affinity and mediates firm lymphocyte adhesion (Chen J et al (2003). Nat Struct Biol. 10, 995-1001).
  • the antibody can either be blocking or non- blocking based upon the desired effect.
  • the antibody is a non-blocking MAdCAM antibody.
  • the antibody is a blocking MAdCAM antibody.
  • Example 6 One non-limiting example of demonstrating whether an antibody is blocking or non-blocking can be found in Example 6, but any method can be used.
  • Each of the references described herein are incorporated by reference in its entirety.
  • the PD-l Agonist is replaced with an IL-2 mutein, such as, but not limited to, the ones described herein.
  • the N-terminus of the homodimer contains two identical Fab domains comprised of two identical light chains, which are separate polypeptides, interfaced with the n-terminal VH-CH1 domains of each heavy chain via the VH/VL interaction and Ckappa or Clambda interaction with CH1.
  • the native disulphide bond between the Ckappa or Clambda with CH1 is present providing a covalent anchor between the light and heavy chains.
  • VH units At the c-terminus of this design are two identical VH units (though non antibody moieties could also be substituted here or at any of the four terminal attachment/fusion points) where by (in this example) the c-terminus of the CH3 domain of the Fc, is followed by a flexible, hydrophilic linker typically comprised of (but not limited to) serine, glycine, alanine, and/or threonine residues, which is followed by a soluble independent VH3 germline family based VH domain. Two such units exist at the c-terminus of this molecule owing to the homodimeric nature centered at the Fc.
  • the N-terminus of the homodimer contains two identical Fab domains comprised of two identical light chains, which, unlike FIG. 3 and FIG. 4, are physically conjoined with the heavy chain at the N-terminus via a linker between the c-terminus of Ckappa or Clambda and the N-terminus of the VH.
  • the linker may be 36-80 amino acids in length and comprised of serine, glycine, alanine and threonine residues.
  • the physically conjoined n-terminal light chains interface with the n-terminal VH-CHl domains of each heavy chain via the VH/VL interaction and Ckappa or Clambda interaction with CH1.
  • the native disulphide bond between the Ckappa or Clambda with CH1 is present providing additional stability between the light and heavy chains.
  • Fab units where by (in this example) the c-terminus of the CH3 domain of the Fc, is followed by a flexible, hydrophilic linker typically comprised of (but not limited to) serine, glycine, alanine, and/or threonine residues, which is followed by a CH1 domain, followed by a VH domain at the c-terminus.
  • the light chain that is designed to pair with the c-terminal CH1/VH domains is expressed as a separate polypeptide, unlike the N-terminal light chain which is conjoined to the n-terminal VH/CH1 domains as described.
  • the C-terminal light chains form an interface at between VH/VL and Ckappa or Clambda with CH1.
  • the native disulphide anchors this light chain to the heavy chain.
  • any of the antibody moieties at any of the four attachment/fusion points can be substituted with a non-antibody moiety, e.g., a effector binding/modulating moiety that does not comprise an antibody molecule.
  • the bispecific antibodies can also be asymmetric as shown in the following non-limiting examples. Non-limiting example are also depicted in FIG. 6, FIG. 7, and FIG. 8, which illustrate an asymmetric/heterodimer approach. Again, in any of these formats, any of the antibody moieties at any of the four attachment/fusion points can be substituted with a non-antibody moiety, e.g., a effector binding/modulating moiety that does not comprise an antibody molecule.
  • the dimerization interface centers around the human IgGl CH2-CH3 domains, which dimerize via a contact interface spanning both CH2/CH2 and CH3/CH3.
  • the heterodimerizing mutations include T366W mutation (kabat) in one CH3 domain and T366S, L368A, and Y407V (kabat) mutations in the other CH3 domain.
  • the heterodimerizing interface may be further stabilized with de novo disulphide bonds via mutation of native residues to cysteine residues such as S354 and Y349 on opposite sides of the CH3/CH3 interface.
  • the resulting bispecific antibodies shown have a total valence comprised of four binding units.
  • the overall molecule can be designed to have bispecificity at just one terminus and monospecificity at the other terminus (trispecificity overall) or bispecificity at either terminus with an overall molecular specificity of 2 or 4.
  • the C-terminus comprises two identical binding domains which could, for example, provide bivalent monospecificity for a tissue tethering target.
  • both binding domains comprise different recognition elements/paratopes and which could achieve recognition of two different epitopes on the same effector moiety target, or could recognize for examples a T cell inhibitory receptor and CD3.
  • the N-terminal binding moieties may be interchanged with other single polypeptide formats such as scFv, single chain Fab, tandem scFv, VH or VHH domain antibody configurations for example.
  • Other types of recognition element may be used also, such as linear or cyclic peptides.
  • FIG. 6 An example of an asymmetric molecule is depicted in FIG. 6.
  • the N- terminus of the molecule is comprised of a first light chain paired with a first heavy chain via VH/VL and Ckappa or Clambda / CH1 interactions and a covalent tether comprised of the native heavy /light chain disulphide bond.
  • a second light chain and a second heavy chain On the opposite side of this heterodimeric molecule at the N- terminus is a second light chain and a second heavy chain which are physically conjoined via a linker between the c-terminus of Ckappa or Clambda and the N-terminus of the VH.
  • the linker may be 36-80 amino acids in length and comprised of serine, glycine, alanine and threonine residues.
  • the physically conjoined n-terminal light chains interface with the n-terminal VH- CH1 domains of each heavy chain via the VH/VL interaction and Ckappa or Clambda interaction with CH1.
  • the native disulphide bond between the Ckappa or Clambda with CH1 is present providing additional stability between the light and heavy chains.
  • an asymmetric molecule can be as illustrated as depicted in FIG.
  • the N-terminus of the molecule is comprised of two different VH3 germlined based soluble VH domains linked to the human IgGl hinge region via a
  • the VH domain connected to the first heavy chain is different to the VH domain connected to the second heavy chain.
  • At the c-terminus of each heavy chain is an additional soluble VH3 germline based VH domain, which is identical on each of the two heavy chains.
  • the heavy chain heterodimerizes via the previously described knobs into holes mutations present at the CH3 interface of the Fc module.
  • an asymmetric molecule can be as illustrated in FIG. 8.
  • This example is similar to the molecule shown in FIG. 7, except both N-terminal Fab units are configured in a way that light chain 1 and light chain 2 are physically conjoined with heavy chain 1 and heavy chain 2 via a linker between the c-terminus of Ckappa or Clambda and the N- terminus of each respective VH.
  • the linker in each case may be 36-80 amino acids in length and comprised of serine, glycine, alanine and threonine residues.
  • the physically conjoined n- terminal light chains interface with the n-terminal VH-CH1 domains of each heavy chain via the VH/VL interaction and Ckappa or Clambda interaction with CH1.
  • the native disulphide bond between the Ckappa or Clambda with CH1 is present providing additional stability between the light and heavy chains.
  • Bi-specific molecules can also have a mixed format. This is illustrated, for example, in FIG. 9, FIG. 10, and FIG. 11.
  • FIG. 9 illustrates a homodimer Fc based approach (see FIGS. 3, 4, and 5), combined with the moiety format selection of FIG. 7, whereby the total molecular valency is four, but specificity is restricted to two specificities.
  • the N-terminus is comprised of two identical soluble VH3 germline based VH domains and the c-terminus is comprised of two identical soluble VH3 germlined based VH domains of different specificity to the N-terminal domains. Therefore, each specificity has a valence of two.
  • any of the antibody moieties at any of the four attachment/fusion points can be substituted with a non-antibody moiety, e.g., an effector binding/modulating moiety that does not comprise an antibody molecule.
  • FIG. 10 illustrates another example.
  • the molecule is comprised of four VH3 germline based soluble VH domains.
  • the first two domains have the same specificity (for example an inhibitory receptor)
  • the 3rd domain from the N-terminus may have specificity for a tissue antigen
  • the fourth domain from the N-terminus may have specificity for human serum albumin (HSA), thereby granting the molecule extended half-life in the absence of an Ig Fc domain.
  • HSA human serum albumin
  • Three glycine, serine, alanine and/or threonine rich linkers exists between domains 1 and 2, domains 2 and 3, and domains 3 and 4.
  • This format may be configured with up to tetraspecificity, but monovalent in each case, or to have bispecificity with bivalency in each case.
  • the order of domains can be changed.
  • any of the antibody moieties can be substituted with a non-antibody moiety, e.g., a effector binding/modulating moiety that does not comprise an antibody molecule.
  • FIG. 11 illustrates yet another approach. This example is similar to FIGS. 3 and 4, in that it is Fc homodimer based with two identical Fab units (bivalent monospecificity) at the N- terminus of the molecule. This example differs in that the C-terminus of each heavy chain is appended with a tandem-scFv.
  • the c-terminus of the CH3 domain of the Fc is linked via a glycine/serine/alanine/threonine based linker to the N-terminus of a first VH domain, which is linked via the C-terminus by a 12-15 amino acid glycine/serine rich linker to the N- terminus of a first VL domain, which linked via a 25-35 amino acid
  • glycine/serine/alanine/threonine based linker at the c-terminus to the N-terminus of a second VH domain which is linked via the c-terminus with a 12-15 amino acid glycine/serine based linker to the N-terminus of a 2nd VL domain.
  • this Fc homodimer based molecule there are therefore two identical tandem scFvs at the c-terminus of the molecule offering either tetravalency for a single tissue antigen for example or bivalency to two different molecules.
  • This format could also be adapted with a heterodimer Fc core allowing two different tandem-scFvs at the c-terminus of the Fc allowing for monovalent tetraspecificity at the c-terminus while retaining either bivalent monospecificity at the N-terminus or monovalent bispecificity at the N-terminal via usage of single chain Fab configurations as in FIGS. 5, 6, and 7.
  • This molecule can therefore be configured to have 2, 3, 4, 5, or 6 specificities.
  • the domain order of scFvs within the tandem— scFv units may be configured to be from N to C terminus either VH-Linker-VL or VL-Linker- VH.
  • any of the antibody moieties at any of the four attachment/fusion points can be substituted with a non-antibody moiety, e.g., a effector binding/modulating moiety that does not comprise an antibody molecule.
  • Bi-specific antibodies can also be constructed to have, for example, shorter systemic PK while having increased tissue penetration.
  • These types of antibodies can be based upon, for example, a human VH3 based domain antibody format. These are illustrated, for example, in FIGS. 12, 13, and 14.
  • FIGS. 12, 13, and 14 each comprised a soluble VH3 germline family based VH domain modules. Each domain is approximately 12.5 kDa allowing for a small overall MW, which, without being bound to any particular theory, should be beneficial for enhanced tissue penetration.
  • none of the VH domains recognize any half-life extending targets such as FcRn or HSA. As illustrated in FIG.
  • the molecule is comprised of two VH domains joined with a flexible hydrophilic gly cine/ serine based linker between the C-terminus of the first domain and N-terminus of the second domain.
  • one domain may recognize a T cell co-stimulatory receptor and the second may recognize a tissue tethering antigen.
  • the molecule is comprised of three VH domains with N-C terminal linkages of hydrophilic gly cine/ serine based linkers.
  • the molecule may be configured to be trispecific but monovalent for each target. It may be bispecific with bivalency for one target and monovalency for another. As illustrated in FIG.
  • the molecule is comprised of four VH domains with N-C terminal Glycine/Serine rich linkers between each domain.
  • This molecule may be configured to be tetraspecific, trispecific, or bispecific with varying antigenic valencies in each case.
  • any of the antibody moieties at can be substituted with a non antibody moiety, e.g., a effector binding/modulating moiety that does not comprise an antibody molecule.
  • FIGs. 15 and 16 are comprised of the naturally heterodimerizing core of the human IgG CHl/Ckappa interface, including the c-terminal heavy /light disulphide bond which covalently anchors the interaction. This format does not contain an Fc or any moieties for half life extension. As illustrated in FIG.
  • the molecule, at the N-terminus of the constant kappa domain is appended with an scFv fragment consisting of an N-terminal VH domain, linked at its C-terminus to the N- terminus of a VL domain via a 12-15 amino acid gly/ser based linker, which is linked by its C- terminus to the N-terminus of the constant kappa domain via the native VL-Ckappa elbow sequence.
  • the CH1 domain is appended at the N-terminus with an scFv fragment consisting of an N-terminal VL domain linked at its c-terminus via a 12-15 amino acid gly/ser linker to the N- terminus of a VH domain, which is linked at its c-terminus to the N-terminus of the CH1 domains via the natural VH-CHl elbow sequence.
  • the molecule has the same N-terminal configuration to Example 13.
  • the C-terminus of the constant kappa and CH1 domains are appended with scFv modules which may be in either the VH-VL or VL-VH configuration and may be either specific for the same antigen or specific for two different antigens.
  • the VH/VL inter-domain linkers may be 12-15 amino acids in length and consisting of gly/ser residues.
  • the scFv binding sub-units may be swapped for soluble VH domains, or peptide recognition elements, or even tandem-scFv elements.
  • This approach can also be configured to use variable lambda and/or constant lambda domains.
  • any of the antibody moieties at any of the attachment/fusion points can be substituted with a non- antibody moiety, e.g., a effector binding/modulating moiety that does not comprise an antibody molecule.
  • FIG. 17 illustrates another embodiment.
  • FIG. 17 represents a tandem scFv format consisting of a first N-terminal VL domain linked at its C-terminus to the N-terminus of a first VH domain with a 12-15 amino acid gly/ser rich linker, followed at the first VH c-terminus by a 25-30 amino acid gly/ser/ala/thr based linker to the N-terminus of a second VL domain.
  • the second VL domain is linked at the C-terminus to the N-terminus of a 2nd VH domain by a 12-15 amino acid gly/ser linker.
  • Each scFv recognizes a different target antigen such as a co stimulatory T cell molecule and a tissue tethering target.
  • a target antigen such as a co stimulatory T cell molecule and a tissue tethering target.
  • any of the antibody moieties can be substituted with a non-antibody moiety, e.g., a effector
  • binding/modulating moiety that does not comprise an antibody molecule.
  • FIG. 18 illustrates another embodiment.
  • FIG. 18 is a F(ab’)2 scFv fusion. This consists of two identical Fab components joined via two disulphide bonds in the native human IgGl hinge region c-terminal of the human IgG CH1 domain. The human IgGl CH2 and CH3 domains are absent. At the c-terminus of heavy chains 1 and 2 are two identical scFv fragments linked via a gly/ser/ala/thr rich linker to the c-terminus of the huIgGl hinge region.
  • the VH is N-terminal in each scFv unit and linked bia a 12-15 amino acid gly/ser rich linker to the N-terminus of a VL domain.
  • An alternative configuration would be N- term- VL-Linker-VH-C-term.
  • the construct is bispecific with bivalency for reach target.
  • any of the antibody moieties at any of the four attachment/fusion points can be substituted with a non-antibody moiety, e.g., a effector binding/modulating moiety that does not comprise an antibody molecule.
  • CD39 molecule refers to a polypeptide having sufficient CD39 sequence that, as part of a therapeutic compound, it phosphohydrolyzes ATP to AMP.
  • a CD39 molecule phosphohydrolizes ATP to AMP equivalent to, or at least, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95% of the rate of a naturally occurring CD39, e.g., the CD39 from which the CD39 molecule was derived.
  • a CD39 molecule has at least 60, 70, 80, 90, 95, 99, or 100% sequence identity ,or substantial sequence identity, with a naturally occurring CD39. Any functional isoform can be used (with CD39 or other proteins discussed herein).
  • Exemplary CD39 sequence include Genbank accession # NP 001767.3 or a mature form from the following sequence:
  • a CD39 molecule comprises a soluble catalytically active form of CD39 found to circulate in human or murine serum, see, e.g., Metabolism of circulating ADP in the bloodstream is mediated via integrated actions of soluble adenylate kinase-l and
  • NTPDasel/CD39 activities Yegutkin et al. FASEB J. 2012 Sep; 26(9):3875-83.
  • a soluble recombinant CD39 fragment is also described in Inhibition of platelet function by recombinant soluble ecto-ADPase/CD39, Gayle, et al., J Clin Invest. 1998 May 1; 101(9): 1851-1859.
  • CD73 molecule refers to a polypeptide having sufficient CD73 sequence that, as part of a therapeutic compound, it dephosphorylates extracellular AMP to adenosine.
  • a CD73 molecule dephosphorylates extracellular AMP to adenosine equivalent to, or at least, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95% of the rate of a naturally occurring CD73, e.g., the CD73 from which the CD73 molecule was derived.
  • a CD73 molecule has at least 60, 70, 80, 90, 95, 99, or 100% sequence identity, or substantial sequence identity, with a naturally occurring CD73.
  • Exemplary CD73 sequences include GenBank AAH65937.1 5'-nucleotidase, ecto (CD73) [Homo sapiens] or a mature form from the following sequence,
  • a CD73 molecule comprises a soluble form of CD73 which can be shed from the membrane of endothelial cells by proteolytic cleavage or hydrolysis of the GPI anchor by shear stress
  • a soluble form of CD73 which can be shed from the membrane of endothelial cells by proteolytic cleavage or hydrolysis of the GPI anchor by shear stress
  • Br J Pharmacol 2000; 129: 921-6 for CD73 fucntion see Colgan et ak, Physiological roles for ecto-5’ -nucleotidase (CD73), Purinergic Signalling, June 2006, 2:351.
  • Cell surface molecule binder refers to a molecule, typically a polypeptide, that binds, e.g., specifically, to a cell surface molecule on a cell, e.g., an
  • the cell surface binder has sufficient sequence from a naturally occurring ligand of the cell surface molecule, that it can specifically bind the cell surface molecule (a cell surface molecule ligand).
  • the cell surface binding is an antibody molecule that binds, e.g., specifically binds, the cell surface molecule.
  • Donor specific targeting moiety refers to a moiety, e.g., an antibody molecule, that as a component of a therapeutic compound, localizes the therapeutic compound preferentially to an implanted donor tissue, as opposed to tissue of a recipient.
  • the donor specific targeting moiety provides site-specific immune privilege for a transplant tissue, e.g., an organ, from a donor.
  • a donor specific targeting moiety it binds to the product, e.g., a polypeptide product, of an allele present at a locus, which allele is not present at the locus in the (recipient) subject. In some embodiments, a donor specific targeting moiety binds to an epitope on product, which epitope is not present in the (recipient) subject.
  • a donor specific targeting moiety as a component of a therapeutic compound, preferentially binds to a donor target or antigen, e.g., has a binding affinity for the donor target that is greater for donor antigen or tissue, e.g., at least 2, 4, 5, 10, 50, 100, 500,
  • a donor specific targeting moiety has a binding affinity for a product of an allele of a locus present in donor tissue (but not present in the subject) at least 2, 4, 5, 10, 50,
  • Affinity of a therapeutic compound of which the donor specific moiety is a component can be measured in a cell suspension, e.g., the affinity for suspended cells having the allele is compared with its affinity for suspended cells not having the allele.
  • the binding affinity for the donor allele cells is below lOnM. In some embodiments, the binding affinity for the donor allele cells is below 100 pM, 50 pM, or 10 pM.
  • the specificity for a product of a donor allele is sufficient that when the donor specific targeting moiety is coupled to an immune-down regulating effector: i) immune attack of the implanted tissue, e.g., as measured by histological inflammatory response, infiltrating T effector cells, or organ function, in the clinical setting - e.g. creatinine for the kidney, is substantially reduced, e.g., as compared to what would be seen in an otherwise similar implant but lacking the donor specific targeting moiety is coupled to an immune-down regulating effector; and/or ii) immune function in the recipient, outside or away from the implanted tissue, is substantially maintained.
  • peripheral blood lymphocyte counts are not substantially impacted, e.g., the level of T cells is within 25, 50, 75, 85, 90, or 95 % of normal, the level of B cells is within 25, 50, 75, 85, 90, or 95 % of normal, and/or the level of
  • granuloctyes (PMNs) cells is within 25, 50, 75, 85, 90, or 95 % of normal, or the level of monocytes is within 25, 50, 75, 85, 90, or 95 % of normal; at therapeutic levels of therapeutic compound, the ex vivo proliferative function of PBMCs (peripheral blood mononuclear cells) against non-disease relevant antigens is substantially normal or is within 70, 80, or 90% of normal; at therapeutic levels of therapeutic compound, the incidence or risk of risk of
  • the donor specific targeting moiety comprises an antibody molecule, a target specific binding polypeptide, or a target ligand binding molecule.
  • Effector refers to an entity, e.g., a cell or molecule, e.g., a soluble or cell surface molecule, which mediates an immune response.
  • Effector ligand binding molecule refers to a polypeptide that has sufficient sequence from a naturally occurring counter-ligand of an effector, that it can bind the effector with sufficient specificity that it can serve as an effector binding/modulating molecule.
  • it binds to effector with at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95% of the affinity of the naturally occurring counter-ligand. In some embodiments, it has at least 60, 70, 80, 90, 95, 99, or 100% sequence identity ,or substantial sequence identity, with a naturally occurring counter-ligand for the effector.
  • Effector specific binding polypeptide refers to a polypeptide that can bind with sufficient specificity that it can serve as an effector binding/modulating moiety.
  • a specific binding polypeptide comprises a effector ligand binding molecule.
  • Elevated risk refers to the risk of a disorder in a subject, wherein the subject has one or more of a medical history of the disorder or a symptom of the disorder, a biomarker associated with the disorder or a symptom of the disorder, or a family history of the disorder or a symptom of the disorder.
  • Functional antibody molecule to an effector or inhibitory immune checkpoint molecule refers to an antibody molecule that when present as the ICIM binding/modulating moiety of a multimerized therapeutic compound, can bind and agonize the effector or inhibitory immune checkpoint molecule.
  • the anti-effector or inhibitory immune checkpoint molecule antibody molecule when binding as a monomer (or binding when the therapeutic compound is not multimerized), to the effector or inhibitory immune checkpoint molecule, does not antagonize, substantially antagonize, prevent binding, or prevent substantial binding, of an endogenous counter ligand of the inhibitory immune checkpoint molecule molecule to inhibitory immune checkpoint molecule.
  • the anti- effector or inhibitory immune checkpoint molecule antibody molecule when binding as a monomer (or binding when the therapeutic compound is not multimerized), to the inhibitory immune checkpoint molecule does not agonize or substantially agonize, the effector or inhibitory molecule.
  • ICIM binding/modulating moiety refers to an effector binding/modulating moiety that, as part of a therapeutic compound, binds and agonizes a cell surface inhibitory molecule, e.g., an inhibitory immune checkpoint molecule, e.g., PD-l, , or binds or modulates cell signaling, e.g., binds a FCRL, e.g., FCRL1-6, or binds and antagonizes a molecule that promotes immune function.
  • a cell surface inhibitory molecule e.g., an inhibitory immune checkpoint molecule, e.g., PD-l, , or binds or modulates cell signaling, e.g., binds a FCRL, e.g., FCRL1-6, or binds and antagonizes a molecule that promotes immune function.
  • IIC binding/modulating moiety refers to an effector binding/modulating moiety that, as part of a therapeutic compound, binds an immunosuppressive immune cell.
  • the IIC binding/modulating moiety increases the number or concentration of an immunosuppressive immune cell at the binding site.
  • ICSM binding/modulating moiety refers to an effector binding/modulating moiety that antagonizes an immune stimulatory effect of a stimulatory, e.g., co-stimulatory, binding pair.
  • a stimulatory or co-stimulatory binding pair comprises two members, 1) a molecule on the surface of an immune cell; and 2) the binding partner for that cell molecule, which may be an additional immune cell, or a non- immune cell.
  • the member on the immune cell surfaces stimulates the immune cell, e.g., a
  • an ICSM binding/modulating moiety binds and antagonizes the immune cell expressed member of a binding pair. For example, it binds and antagonizes 0X40.
  • an ICSM binding/modulating moiety binds and antagonizes the member of the binding pair that itself binds the immune cell expressed member, e.g., it binds and antagonizes OX40L. In either case, inhibition of stimulation or co-stimulation of an immune cell is achieved.
  • the ICSM binding/modulating moiety decreases the number or the activity of an immunostimulating immune cell at the binding site.
  • IL-2 mutein molecule refers to an IL2 variant that binds with high affinity to the CD25 (IL-2R alpha chain) and with low affinity to the other IL-2R sigalling components CD122 (IL-2R beta) and CD132 (IL-2R gamma).
  • Such an IL-2 mutein molecule preferentially activates Treg cells.
  • an IL-2 mutein activates Tregs at least 2, 5, 10, or 100 fold more than cytotoxic or effector T cells.
  • Exemplary IL-2 mutein molecules are described in
  • an IIC binding/modulating moiety comprises an IL-2 mutein, or active fragment thereof, coupled, e.g., fused, to another
  • polypeptide e.g., a polypeptide that extends in vivo half life, e.g., an immunoglobulin constant region, or a multimer or dimer thereof, e.g., AMG 592.
  • the therapeutic compound comprises the IL-2 portion of AMG 592.
  • the therapeutic compound comprises the IL-2 portion but not the immunoglobulin portion of AMG 592.
  • the mutein does not comprise a Fc region.
  • the muteins are engineered to contain a Fc region because such region has been shown to increase the half-life of the mutein.
  • the extended half-life is not necessary for the methods described and embodied herein.
  • the Fc region that is fused with the IL-2 mutein comprises a N297 mutations, such as, but not limited to, N297A. In some embodiments, the Fc region that is fused with the IL-2 mutein does not comprise a N297 mutation, such as, but not limited to, N297A.
  • the inhibitory immune checkpoint molecule ligand molecule when binding as a monomer (or binding when the therapeutic compound is not multimerized), to its cognate ligand, e.g., PD-l, does not antagonize or substantially antagonize, or prevent binding, or prevent substantial binding, of an endogenous inhibitory immune checkpoint molecule ligand to the inhibitory immune checkpoint molecule.
  • the PD-L1 molecule does not antagonize binding of endogenous PD- Ll to PD-l.
  • the inhibitory immune checkpoint molecule ligand when binding as a monomer, to its cognate inhibitory immune checkpoint molecule does not agonize or substantially agonize the inhibitory immune checkpoint molecule.
  • a PD-L1 molecule when binding to PD-l does not agonize or substantially agonize PD-l .
  • an inhibitory immune checkpoint molecule ligand molecule has at least 60, 70, 80, 90, 95, 99, or 100% sequence identity ,or substantial sequence identity, with a naturally occurring inhibitory immune checkpoint molecule ligand.
  • inhibitory immune checkpoint molecule ligand molecules include: a PD-L1 molecule, which binds to inhibitory immune checkpoint molecule PD-l, and in embodiments has at least 60, 70, 80, 90, 95, 99, or 100% sequence identity ,or substantial sequence identity, with a naturally occurring PD-L1, e.g., the PD-L1 molecule comprising the sequence of
  • the active fragment comprises residues 19 to 290 of the PD-L1 sequence; ; a HLA-G molecule, which binds to any of inhibitory immune checkpoint molecules KIR2DL4, LILRB1, and LILRB2, and in embodiments has at least 60, 70
  • MVVMAPRTLFLLL SGALTLTETW AGSHSMRYF S AAV SRPGRGEPRFIAMGYVDDTQF V RFD SD S ACPRMEPRAPWVEQEGPEYWEEETRNTK AHAQTDRMNLQTLRGYYNQ SE AS SHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCE AANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRADPPKTHVTHHPVFDYEATLRCW ALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQ HEGLPEPLMLRWKQ S SLPTIPIMGI V A (SEQ ID NO: 4).
  • Inhibitory molecule counter ligand molecule refers to a polypeptide having sufficient inhibitory molecule counter ligand sequence such that when present as the ICIM binding/modulating moiety of a multimerized therapeutic compound, can bind and agonize a cognate inhibitory molecule.
  • the inhibitory molecule counter ligand molecule when binding as a monomer (or binding when the therapeutic compound is not multimerized), to the inhibitory molecule, does not antagonize, substantially antagonize, prevent binding, or prevent substantial binding, of an endogenous counter ligand of the inhibitory molecule to the inhibitory molecule.
  • the inhibitory molecule counter ligand molecule when binding as a monomer (or binding when the therapeutic compound is not multimerized), to the inhibitory molecule, does not agonize or substantially agonize, the inhibitory molecule.
  • amino acid sequence the term “substantially identical” is used herein to refer to a first amino acid that contains a sufficient or minimum number of amino acid residues that are i) identical to, or ii) conservative substitutions of aligned amino acid residues in a second amino acid sequence such that the first and second amino acid sequences can have a common structural domain and/or common functional activity.
  • amino acid sequences that contain a common structural domain having at least about 85%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a reference sequence, e.g., a sequence provided herein.
  • nucleotide sequence in the context of nucleotide sequence, the term "substantially identical" is used herein to refer to a first nucleic acid sequence that contains a sufficient or minimum number of nucleotides that are identical to aligned nucleotides in a second nucleic acid sequence such that the first and second nucleotide sequences encode a polypeptide having common functional activity, or encode a common structural polypeptide domain or a common functional polypeptide activity.
  • “functional variant” refers to polypeptides that have a substantially identical amino acid sequence to the naturally-occurring sequence, or are encoded by a substantially identical nucleotide sequence, and are capable of having one or more activities of the naturally- occurring sequence.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
  • the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence.
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the percent identity between two amino acid sequences is determined using the Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453 ) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
  • the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6.
  • a particularly preferred set of parameters are a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
  • the percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4: 11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • nucleic acid and protein sequences described herein can be used as a "query sequence" to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10.
  • Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25:3389-3402.
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • XBLAST and NBLAST See http://www.ncbi.nlm.nih.gov.
  • hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions describes conditions for hybridization and washing.
  • amino acid is intended to embrace all molecules, whether natural or synthetic, which include both an amino functionality and an acid functionality and capable of being included in a polymer of naturally-occurring amino acids.
  • exemplary amino acids include naturally-occurring amino acids; analogs, derivatives and congeners thereof; amino acid analogs having variant side chains; and all stereoisomers of any of any of the foregoing.
  • amino acid includes both the D- or L- optical isomers and peptidomimetics.
  • a “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • CD39 molecule CD39 molecule, a CD73 molecule, a Cell surface molecule binder, Donor specific targeting moiety Effector ligand binding molecule, ICIM binding/modulating moiety IIC binding/modulating moiety, an inhibitory immune checkpoint molecule ligand molecule, Inhibitory molecule counter ligand molecule, SM binding/modulating moiety, or ICSM binding/modulating moiety.
  • SM binding/modulating moiety refers to an effector binding/modulating moiety that, as part of a therapeutic compound, promotes an immuno- suppressive local microenvironment, e.g., by providing in the proximity of the target, a substance that inhibits or minimizes attack by the immune system of the target.
  • the SM binding/modulating moiety comprises, or binds, a molecule that inhibits or minimizes attack by the immune system of the target.
  • a therapeutic compound comprises an SM binding/modulating moiety that binds and accumulates a soluble substance, e.g., an endogenous or exogenous substance, having immunosuppressive function.
  • a therapeutic compound comprises an SM binding/modulating moiety that binds and inhibits, sequesters, degrades or otherwise neutralizes a substance, e.g., a soluble substance, typically and endogenous soluble substance, that promotes immune attack.
  • a therapeutic compound comprises an SM binding/modulating moiety that comprises an immune-suppressive substance, e.g. a fragment of protein known to be
  • an effector molecule binding moiety that binds, or comprises, a substance e.g., a CD39 molecule or a CD73 molecule, that depletes a component, that promotes immune effector cell function, e.g., ATP or AMP.
  • Specific targeting moiety refers to donor specific targeting moiety or a tissue specific targeting moiety.
  • Subject refers to a mammalian subject, e.g., a human subject.
  • the subject is a non-human mammal, e.g., a horse, dog, cat, cow, goat, or Pig ⁇
  • Target ligand binding molecule refers to a polypeptide that has sufficient sequence from a naturally occurring counter-ligand of a target ligand that it can bind the target ligand on a target tissue (e.g., donor tissue or subject target tissue) with sufficient specificity that it can serve as a specific targeting moiety. In some embodiments, it binds to target tissue or cells with at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95% of the affinity of the naturally occurring counter-ligand. In some embodiments, it has at least 60, 70, 80, 90, 95, 99, or 100% sequence identity ,or substantial sequence identity, with a naturally occurring counter-ligand for the target ligand.
  • Target site refers to a site which contains the entity, e.g., epitope, bound by a targeting moiety.
  • the target site is the site at which immune privilege is established.
  • Tissue specific targeting moiety refers to a moiety, e.g., an antibody molecule, that as a component of a therapeutic molecule, localizes the therapeutic molecule preferentially to a target tissue, as opposed to other tissue of a subject.
  • the tissue specific targeting moiety provides site-specific immune privilege for a target tissue, e.g., an organ or tissue undergoing or at risk for autoimmune attack.
  • a tissue specific targeting moiety binds to a product, e.g., a polypeptide product, which is not present outside the target tissue, or is present at sufficiently low levels that, at therapeutic concentrations of therapeutic molecule, unacceptable levels of immune
  • tissue specific targeting moiety binds to an epitope, which epitope is not present outside, or not substantially present outside, the target site.
  • a tissue specific targeting moiety as a component of a therapeutic compound, preferentially binds to a target tissue or target tissue antigen, e.g., has a binding affinity for the target tissue or antigen that is greater for target antigen or tissue, e.g., at least 2, 4, 5, 10, 50, 100, 500, 1,000, 5,000, or 10,000 fold greater, than its affinity for than for non-target tissue or antigen present outside the target tissue.
  • Affinity of a therapeutic compound of which the tissue specific moiety is a component can be measured in a cell suspension, e.g., the affinity for suspended cells having the target antigen is compared with its affinity for suspended cells not having the target antigen.
  • the binding affinity for the target antigen bearing cells is below lOnM.
  • the binding affinity for the target antigen bearing cells is below 100 pM, 50 pM, or 10 pM.
  • the specificity for a target antigen is sufficient, that when the tissue specific targeting moiety is coupled to an immune-down regulating effector: i) immune attack of the target tissue, e.g., as measured by histological inflammatory response, infiltrating T effector cells, or organ function, in the clinical setting - e.g.
  • creatinine for kidney is substantially reduced, e.g., as compared to what would be seen in an otherwise similar implant but lacking the tissue specific targeting moiety is coupled to an immune-down regulating effector; and/or ii) immune function in the recipient, outside or away from the target tissue, is substantially maintained.
  • one or more of the following is seen: at therapeutic levels of therapeutic compound, peripheral blood lymphocyte counts are not substantially impacted, , e.g., the level of T cells is within 25, 50, 75, 85, 90, or 95 % of normal, the level of B cells is within 25, 50, 75, 85, 90, or 95 % of normal, and/or the level of granulocytes (PMNs) cells is within 25, 50, 75, 85, 90, or 95 % of normal, or the level of monocytes is within 25, 50, 75, 85, 90, or 95 % of normal 1; at therapeutic levels of therapeutic compound, the ex vivo proliferative function of PBMCs (peripheral blood mononuclear cells) against non-disease relevant antigens is substantially normal or is within 70, 80, or 90% of normal; at therapeutic levels of therapeutic compound, the incidence or risk of risk of opportunistic infections and cancers associated with immunosuppression is not substantially increased over normal; or at therapeutic levels of therapeutic compound, the incidence or
  • the tissue specific targeting moiety comprises an antibody molecule.
  • the donor specific targeting moiety comprises an antibody molecule, a target specific binding polypeptide, or a target ligand binding molecule.
  • the tissue specific targeting moiety binds a product, or a site on a product, that is present or expressed exclusively, or substantially exclusively, on target tissue.
  • Methods and compounds described herein provide for a therapeutic compound having an effector binding/modulating moiety comprising an ICIM binding/modulating moiety, that directly binds and activates an inhibitory receptor on the surface of an immune cell, e.g., to reduce or eliminate, or substantially eliminate, the ability of the immune cell to mediate immune attack.
  • Coupling of the ICIM binding/modulating moiety to a targeting entity promotes site- specific or local down regulation of the immune cell response, e.g., confined substantially to the locations having binding sites for the targeting moiety.
  • normal systemic immune function is substantially retained.
  • an ICIM binding/modulating moiety comprises an inhibitory immune checkpoint molecule counter ligand molecule, e.g., a natural ligand, or fragment of a natural ligand (e.g., PD-L1 or HLA-G) of the inhibitory immune checkpoint molecule.
  • an ICIM binding/modulating moiety comprises a functional antibody molecule, e.g., a functional antibody molecule comprising an scFv binding domain, that engages inhibitory immune checkpoint molecule.
  • the ICIM binding/modulating moiety comprising, e.g., a functional antibody molecule, or inhibitory immune checkpoint molecule ligand molecule, binds the inhibitory receptor but does not prevent binding of a natural ligand of the inhibitory receptor to the inhibitory receptor.
  • a targeting moiety is coupled, e.g., fused, to an ICIM binding/modulating moiety, comprising, e.g., an scFv domain, and configured so that upon binding of an inhibitory receptor while in solution (e.g., in blood or lymph) (and presumably in a monomeric format), the therapeutic molecule: i) fails to agonize, or fails to substantially agonize (e.g., agonizes at less than 30, 20, 15, 10, or 5% of the level seen with a full agonizing molecule) the inhibitory receptor on the immune cell; and/or ii) fails to antagonize, or fails to substantially antagonize (e.g., antagonizes at less than 30, 20, 15, 10, or 5% of the level seen with a full antagonizing molecule) the inhibitory receptor on the immune cell.
  • ii fails to agonize, or fails to substantially agonize (e.g., agonizes at less than 30, 20, 15, 10, or 5% of the level
  • a candidate molecule can be evaluated for its ability to agonize or not agonize by its ability to either increase or decrease the immune response in an in vitro cell based assay wherein the target is not expressed, e.g., using an MLR-based assay (mixed lymphocyte reaction).
  • candidate ICIM binding/modulating moieties can reduce, completely or substantially eliminate systemic immunosuppression and systemic immune activation.
  • the targeting domain of the therapeutic compound, when bound to target will serve to cluster or multimerize the therapeutic compound on the surface of the tissue desiring immune protection.
  • the ICIM binding/modulating moiety e.g., an ICIM binding/modulating moiety comprising a scFv domain, requires a clustered or multimeric state to be able to deliver an agonistic and immunosuppressive signal, or substantial levels of such signal, to local immune cells.
  • This type of therapeutic can, for example, provide to a local immune suppression whilst leaving the systemic immune system unperturbed or substantially unperterbed. That is, the immune suppression is localized to where the suppression is needed as opposed to being systemic and not localized to a particular area or tissue type.
  • the therapeutic compound upon binding to the target e.g., a target organ, tissue or cell type, the therapeutic compound coats the target, e.g., target organ, tissue or cell type.
  • the target e.g., target organ, tissue or cell type.
  • this therapeutic will provide an‘off signal only at, or to a greater extent at, the site of therapeutic compound accumulation.
  • a candidate therapeutic compound can be evaluated for the ability to bind, e.g., specifically bind, its target, e.g., by ELISA, a cell based assay, or surface plasm on resonance by. This property should generally be maximized, as it mediates the site-specificity and local nature of the immune privilege.
  • a candidate therapeutic compound can be evaluated for the ability to down regulate an immune cell when bound to target, e.g., by a cell based activity assay. This property should generally be maximized, as it mediates the site-specificity and local nature of the immune privilege.
  • the level of down regulation effected by a candidate therapeutic compound in monomeric (or non-bound) form can be evaluated, e.g., by a cell based activity assay.
  • This property should generally be minimized, as could mediate systemic down regulation of the immune system.
  • the level of antagonism of a cell surface inhibitory molecule, e.g., an inhibitory immune checkpoint molecule, effected by a candidate therapeutic compound in monomeric (or non-bound) form can be evaluated , e.g.,by , e.g., by a cell based activity assay.
  • This property should generally be minimized, as could mediate systemic unwanted activation of the immune system.
  • the properties should be selected and balanced to produce a sufficiently robust site specific immune privilege without unacceptable levels of non-site specific agonism or antagonism of the inhibitory immune checkpoint molecule.
  • inhibitory molecules e.g., an inhibitory immune checkpoint molecule
  • Table 1 This table lists molecules to which exemplary ICIM binding moieties can bind.
  • Programmed cell death protein 1 (often referred to as PD-l) is a cell surface receptor that belongs to the immunoglobulin superfamily. PD-l is expressed on T cells and other cell types including, but not limited to, B cells, myeloid cells, dendritic cells, monocytes, T regulatory cells, iNK T cells. PD-l binds two ligands, PD-L1 and PD-L2, and is an inhibitory immune checkpoint molecule. Engagement with a cognate ligand, PD-L1 or PD-L2, in the context of engagement of antigen loaded MCH with the T Cell Receptor on a T cell minimizes or prevents the activation and function of T cells.
  • the inhibitory effect of PD-l can include both promoting apoptosis (programmed cell death) in antigen specific T-cells in lymph nodes and reducing apoptosis in regulatory T cells (suppressor T cells).
  • a therapeutic compound comprises an ICIM binding/modulating moiety which agonizes PD-l inhibition.
  • An ICIM binding/modulating moiety can include an inhibitory molecule counter ligand molecule, e.g., comprising a fragment of a ligand of PD-l (e.g., a fragment of PD-L1 or PD-L2) or another moiety, e.g., a functional antibody molecule, comprising, e.g., an scFv domain that binds PD-l.
  • an inhibitory molecule counter ligand molecule e.g., comprising a fragment of a ligand of PD-l (e.g., a fragment of PD-L1 or PD-L2) or another moiety, e.g., a functional antibody molecule, comprising, e.g., an scFv domain that binds PD-l.
  • a therapeutic compound comprises a targeting moiety that is preferentially binds a donor antigen not present in, present in substantially lower levels in the subject, e.g., a donor antigen from Table 2, and is localized to donor graft tissue in a subject. In some embodiments, it does not bind, or does not substantially bind, other tissues.
  • a therapeutic compound can include a targeting moiety that is specific for HLA- A2 and specifically binds donor allograft tissue but does not bind, or does not substantially bind, host tissues.
  • the therapeutic compound comprises an ICIM
  • binding/modulating moiety e.g., an inhibitory molecule counter ligand molecule, e.g., comprising a fragment of a ligand of PD-l (e.g., a fragment of PD-L1 or PD-L2) or another moiety, e.g., a functional antibody molecule, comprising, e.g., an scFv domain that binds PD-l, such that the therapeutic compound, e.g., when bound to target, activates PD-l .
  • the therapeutic compound targets an allograft and provides local immune privilege to the allograft.
  • a therapeutic compound comprises a targeting moiety that is preferentially binds to an antigen of Table 3, and is localized to the target in a subject, e.g., a subject having an autoimmune disorder, e.g., an autoimmune disorder of Table 3. In some embodiments, it does not bind, or does not substantially bind, other tissues.
  • a targeting moiety that is preferentially binds to an antigen of Table 3, and is localized to the target in a subject, e.g., a subject having an autoimmune disorder, e.g., an autoimmune disorder of Table 3. In some embodiments, it does not bind, or does not substantially bind, other tissues.
  • the therapeutic compound comprises an ICIM binding/modulating moiety, e.g., an inhibitory molecule counter ligand molecule, e.g., comprising a fragment of a ligand of PD-l (e.g., a fragment of PD-L1 or PD-L2) or another moiety, e.g., a functional antibody molecule, comprising, e.g., an scFv domain that binds PD-l, such that the therapeutic compound, e.g., when bound to target, activates PD-l .
  • the therapeutic compound targets a tissue subject to autoimmune attack and provides local immune privilege to the tissue.
  • PD-L1 and PDL2, or polypeptides derived therefrom can provide candidate ICIM binding moieties.
  • this molecule in monomer form, e.g., when the therapeutic compound is circulating in blood or lymph, this molecule could have an undesired effect of antagonizing the PD-L1/PD-1 pathway, and may only agonize the PD-l pathway when clustered or multimerized on the surface of a target, e.g., a target organ.
  • a therapeutic compound comprises an ICIM binding/modulating moiety comprising a functional antibody molecule, e.g., a scFv domain, that is inert, or substantially inert, to the PD-l pathway in a soluble form but whch agonizes and drives an inhibitory signal when multimerized (by the targeting moiety) on the surface of a tissue.
  • a functional antibody molecule e.g., a scFv domain
  • KIR2DL4, LILRB1, and LILRB2 are inhibitory molecules found on T cells, NK cells, and myeloid cells.
  • HLA-G is a counter ligand for each.
  • KIR2DL4 is also known as CD158D, G9P, KIR-103AS, KIR103, KIR103AS, KIR, KIR- 2DL4, killer cell immunoglobulin like receptor, and two Ig domains and long cytoplasmic tail 4.
  • LILRB1 is also known as LILRB1, CD85J, ILT-2, ILT2, LIR-l, LIR1, MIR-7, MIR7, PIR-B, PIRB, leukocyte immunoglobulin like receptor Bl.
  • LILRB2 is also known as CD85D, ILT-4, LIR-2, LIR2, MIR- 10, MIR10, and ILT4.
  • a therapeutic compound comprising an HLA-G molecule can be used to provide inhibitory signals to an immune cell comprising any of KIR2DL4, LILRB1, and LILRB2, e.g., with multimerized therapeutic compound molecules comprising an HLA-G molecule and thus provide site-specific immune privilege.
  • a therapeutic compound comprising an agonistic anti-KIR2DL4, anti-LILRBl, or anti- LILRB2 antibody molecule can be used to provide inhibitory signals to an immune cell comprising any of KIR2DL4, LILRB1, and LILRB2.
  • HLA-G only delivers an inhibitory signal when multimerized, for example, when expressed on the surface of a cell or when conjugated to the surface of a bead.
  • a therapeutic compound comprising an HLA-G molecule which therapeutic compound does not multimerize in solution (or does not multimerize sufficiently to result in significant levels of inhibitory molecule agonization), is provided.
  • the use of HLA-G molecules that minimize mulitmerization in solution will minimize systemic agonization of immune cells and unwanted immune suppression.
  • HLA-G is not effective in down regulation unless multimerized, that binding of the therapeutic compound to target, through the targeting moiety, multimerizes the ICIM binding entity, and that the multimerized ICIM binding entity, binds and clusters inhibitory molecules on the surface of an immune cell, thus mediating a negative signal that down regulates the immune cell.
  • infiltrating immune cells attempting to damage the target tissue including antigen presenting cells and other myeloid cells, NK cells and T cells, are down regulated.
  • HLA-G molecules minimize antagonism when in monomeric form are desirable, the redundancy of LILRB 1 and LILRB2 will minimize, the impact on systemic even with some monomeric antagonism.
  • the therapeutic compound comprises an ICIM binding/modulating moiety that comprises a HLA-G molecule, e.g., an B2M-free isoform (e.g., HLA-G5), see Carosella et al., Advances in lmmunology, 2015, 127:33.
  • HLA-G preferentially binds LILRB2.
  • MVVMAPRTLFLLL SGALTLTETW AGSHSMRYF S AAV SRPGRGEPRFIAMGYVDDTQF V RFD SD S ACPRMEPRAPWVEQEGPEYWEEETRNTK AHAQTDRMNLQTLRGYYNQ SE AS SHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCE AANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRADPPKTHVTHHPVFDYEATLRCW ALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQ HEGLPEPLMLRWKQ S SLPTIPIMGI V AGL VVL A A V VT GA A V A VL WRKK S SD (SEQ ID NO: 5).
  • a candidate HLA-G molecule can be tested for suitability for use in methods and compounds, e.g., by methods analogous to those described in“Synthetic HLA-G proteins for therapeutic use in transplantation,” LeMaoult et al., 2013 The FASEB Journal 27:3643.
  • a therapeutic compound comprises a targeting moiety that is preferentially binds a donor antigen not present in, present in substantially lower levels in the subject, e.g., a donor antigen from Table 2, and is localized to donor graft tissue in a subject. In some embodiments, it does not bind, or does not substantially bind, other tissues.
  • a therapeutic compound can include a targeting moiety that is specific for HLA- A2 and specifically binds a donor allograft but does not bind host tissues and is combined with an ICIM binding/modulating moiety that comprises a HLA-G molecule that binds KIR2DL4, LILRB 1 , or LILRB2, such that the therapeutic compound, e.g., when bound to target, activates KIR2DL4, LILRB1, or LILRB2.
  • the therapeutic compound targets an allograft and provides local immune privilege to the allograft.
  • a therapeutic compound comprises a targeting moiety that is preferentially binds a tissue specific antigen, e.g., an antigen from Table 3, and is localized to the target site in a subject, e.g., a subject having an autoimmune disorder, e.g., an anutoimmune disorder from Table 3. In some embodiments, it does not bind, or does not substantially bind, other tissues.
  • the therapeutic compound comprises an ICIM binding/modulating moiety that comprises a HLA-G molecule binds KIR2DL4, LILRB1, or LILRB2, such that the therapeutic compound, e.g., when bound to target, activates KIR2DL4, LILRB1, or LILRB2.
  • the therapeutic compound targets an tissue subject to autoimmune attack and provides local immune privilege to the tissue.
  • HLA-G-B2M fusion protein that can also bind LILRB 1.
  • crystal structure of HLA-G was determined using HLA- G/B2M monomers (Clements et al. 2005 PNAS 102:3360)
  • FCRL1-6 generally inhibit B cell activation or function. These type 1 transmembrane glycoproteins are composed of different combinations of 5 types of immunoglobulin-like domains, with each protein consisting of 3 to 9 domains, and no individual domain type conserved throughout all of the FCRL proteins. In general, FCRL expression is restricted to lymphocytes, with the primary expression in B -lymphocytes. Generally, FCRLs function to repress B-cell activation.
  • An ICIM binding/modulating moiety can comprise an agonistic anti-BCMA antibody molecule.
  • the therapeuticcompound comprises an anti-FCRL antibody molecule and an anti-B cell receptor (BCR) antibody molecule. While not wishing to be bound be theory is is believed that a therapeutic compound comprising anti -body molecules of both specificities will bring the FCRL into close proximity with the BCR and inhibit BCR signaling.
  • Effector binding/modulating moiety can comprise an agonist or antagonist of a butyrophilin.
  • an effector binding/modulating moiety an agonistic or functional BTN1A1 molecule, BTN2A2 molecule, BTNL2 molecule, or BTNL1 molecule.
  • a BTNXi molecule has at least 60, 70, 80, 90, 95, 99, or 100% sequence identity, or substantial sequence identity, with a naturally occurring butyrophilin or butyrophilin-like molecule.
  • an effector binding/modulating moiety an antagonistic BTNL8 molecule.
  • An antagonistic BTNL8 molecule refers to a polypeptide having sufficient BTNL8 sequence that, as part of a therapeutic compound, it inhibits the activation, proliferation, or secretion of cytokine by a resting T cell.
  • a BTNL8 molecule has at least 60, 70, 80, 90, 95, 99, or 100% sequence identity, or substantial sequence identity, with a naturally occurring butyrophilin.
  • IIC BINDING/MODULATING MOIETIES EFFECTOR BINDING/MODULATING MOIETIES THAT RECRUIT IMMUNOSUPPRESSIVE T CELLS
  • a therapeutic compound comprises an effector binding/modulating moiety, e.g., an IIC binding/modulating moiety, that binds, activates, or retains
  • immunosuppressive cells e.g., immunosuppressive T cells
  • the IIC binding/modulating moiety e.g., an IIC binding/modulating moiety comprising an antibody molecule, comprising, e.g., an scFv binding domain
  • immunosuppressive cell types e.g., Tregs, e.g., Foxp3+CD25+ Tregs.
  • Organ, tissue or specific cell type tolerance is associated with an overwhelming increase of Tregs proximal and infiltrating the target organ; in embodiments, the methods and compounds described herein synthetically re-create and mimic this physiological state.
  • an immunosuppressive microenvironment is created that serves to protect the organ of interest from the immune system.
  • GARP-BINDERS AS A TREG AND TGFB TARGETING MOLECULE
  • GARP is a membrane protein receptor for latent TGF-beta expressed on the surface of activated Tregs (Tran et al. 2009 PNAS 106: 13445 and Wang et al. 2009 PNAS 106: 13439).
  • a therapeutic compound comprises an IIC binding entity that binds one or both of soluble GARP and GARP-expressing cells, such as activated human Tregs, and a targeting moiety that targets the therapeutic compound to the target tissue of interest.
  • IIC binding/modulating moieties that comprises a GARP -Binder include, e.g., an IIC
  • an anti-GARP antibody molecule e.g., an anti-GARP scFv domain.
  • the therapeutic compound that comprises a GARP binder effects accumulation of GARP-expressing Tregs at the site targeted by the targeting moiety of the therapeutic compound, e.g., a transplant or site of organ injury.
  • a therapeutic compound that comprises a GARP binder effects can also effect accumulation of soluble GARP at site of organ injury, which will serve to bind and activate TGFB1, an immuno-suppressive cytokine, in a local manner (Fridrich et al. 2016 PLoS One H :e0l53290; doi:
  • an effector binding/modulating moiety that comprises a GARP binder can act as either a IIC
  • binding/modulating moiety or an SM binding/modulating moiety.
  • CTLA4 AS A TREG TARGETING AND T EFFECTOR CELL SILENCING MOLECULE
  • an effector binding/modulating moiety e.g., comprises an antibody molecule, e.g., an scFv domain, that binds CTLA4 expressed on the surface of Tregs.
  • the therapeutic molecule accumulates or retains CTLA4+ Tregs at the target site, with local immunosuppression the consequence.
  • CTLA4 is also expressed on activated T cells.
  • a therapeutic compound comprising an effector binding/modulating moiety e.g., an anti-CTLA4 antibody, or a functional anti-CTLA4 antibody, can down regulate the CTLA4 expressing T cell.
  • an effector binding/modulating moiety e.g., an anti-CTLA4 antibody, or a functional anti-CTLA4 antibody
  • the effector moiety can also act as an ICIM binding/modulating moiety.
  • the anti-CTLA4 binder is neither antagonizing or agonizing when in monomeric format, and is only agonizing when clustered or multimerized upon binding to the target.
  • the binding of the therapeutic compound, via the targeting moiety, to the target effects multimerization of therapeutic compound.
  • CTLA4 bound by the effector binding/modulating moiety of the therapeutic compound is clustered, and an inhibitory signal by engagement of CTLA4 expressed by memory and activated T cells
  • the anti-CTLA4 binder is neither antagonizing or agonizing when in monomeric format, and is only agonizing when clustered or multimerized upon binding to the target.
  • IL-2 MUTEIN MOLECULES IL2 RECEPTOR BINDERS THAT ACTIVATE TREGS
  • IL-2 mutein molecules that preferentially expand or stimulate Treg cells (over cytotoxic T cells) can be used as an IIC binding/modulating moiety.
  • IIC binding/modulating moiety comprises a IL-2 mutein molecule.
  • IL-2 mutein molecule or“IL-2 mutein” refers to an IL-2 variant that preferentially activates Treg cells.
  • an IL-2 mutein molecule activates Tregs at least 2, 5, 10, or 100 fold more than cytotoxic T cells.
  • Treg cells can be found in U.S. Patent No. 9,580,486 at, for example, Examples 2 and 3, or in
  • the immature sequence of IL-2 can be represented by
  • an IIC binding/modulating moiety comprises an IL-2 mutein, or active fragment thereof, coupled, e.g., fused, to another polypeptide, e.g., a polypeptide that extends in vivo half life, e.g., an immunoglobulin constant region, or a multimer or dimer thereof.
  • An IL-2 mutein molecule can be prepared by mutating one or more of the residues of IL- 2.
  • Non-limiting examples of IL-2-muteins can be found in WO2016/164937, US9580486, US7105653, US9616105, US 9428567, US2017/0051029, US2014/0286898A1,
  • the IL-2 mutein molecule comprises a serine substituted for cysteine at position 125 of the mature IL-2 sequence.
  • the cysteine at position 125 is also substituted with a valine or alanine.
  • the IL-2 mutein molecule comprises a V91K substitution.
  • the IL-2 mutein molecule comprises a N88D substitution.
  • the IL-2 mutein molecule comprises a N88R substitution. In some embodiments, the IL-2 mutein molecule comprises a substitution of H16E, D84K, V91N, N88D, V91K, or V91R, any combinations thereof. In some embodiments, these IL-2 mutein molecules also comprise a substitution at position 125 as described herein. In some embodiments, the IL-2 mutein molecule comprises one or more substitutions selected from the group consisting of:
  • the amino acid sequence of the IL-2 mutein molecule differs from the amino acid sequence set forth in mature IL-2 sequence with a C125A or C125S substitution and with one substitution selected from T3N, T3A, L12G, L12K, L12Q L12S, Q13G, E15A, E15G, E15S, H16A, H16D, H16G, H16K,
  • the IL-2 mutein molecule differs from the amino acid sequence set forth in mature IL-2 sequence with a C125A or C125S substitution and with one substitution selected from D20H, D20I, D20Y, D20E, D20G, D20W, D84A, D84S, H16D, H16G, H16K, H16R, H16T, H16V, I92K, I92R, L12K, L19D, L19N, L19T, N88D, N88R, N88S, V91D, V91G, V91K, and V91S.
  • the IL-2 mutein comprises N88R and/or D20H mutations.
  • the IL-2 mutein molecule comprises a mutation in the polypeptide sequence at a position selected from the group consisting of amino acid 30, amino acid 31, amino acid 35, amino acid 69, and amino acid 74.
  • the mutation at position 30 is N30S.
  • the mutation at position 31 is Y31H.
  • the mutation at position 35 is K35R.
  • the mutation at position 69 is V69A.
  • the mutation at position 74 is Q74P.
  • the mutein comprises a V69A mutation, a Q74P mutation, a N88D or N88R mutation, and one or more of L53I, L56I, L80I, or Ll 181 mutations.
  • the mutein comprises a V69A mutation, a Q74P mutation, a N88D or N88R mutation, and a L to I mutation selected from the group consisting of: L53I, L56I, L80I, and Ll 181 mutation.
  • the IL-2 mutein comprises a V69A, a Q74P, a N88D or N88R mutation, and a L53I mutation.
  • the IL-2 mutein comprises a V69A, a Q74P, a N88D or N88R mutation, and a L56I mutation. In some embodiments, the IL-2 mutein comprises a V69A, a Q74P, a N88D or N88R mutation, and a L80I mutation. In some embodiments, the IL-2 mutein comprises a V69A, a Q74P, a N88D or N88R mutation, and a Ll 181 mutation. As provided for herein, the muteins can also comprise a C125A or C125S mutation.
  • the IL-2 mutein molecule comprises a substitution selected from the group consisting of: N88R, N88I, N88G, D20H, D109C, Q126L, Q126F, D84G, or D84I relative to mature human IL-2 sequence provided above.
  • the IL-2 mutein molecule comprises a substitution of D109C and one or both of a N88R substitution and a C125S substitution.
  • the cysteine that is in the IL-2 mutein molecule at position 109 is linked to a polyethylene glycol moiety, wherein the polyethylene glycol moiety has a molecular weight of between 5 and 40 kDa.
  • any of the substitutions described herein are combined with a substitution at position 125.
  • the substitution can be a C125S, C125A, or C125V substitution.
  • the IL-2 mutein has a subsitutiton/mutation at one or more of positions 73, 76, 100, or 138 that correspond to SEQ ID NO: 15 or positions at one or more of positions 53, 56, 80, or 118 that correspond to SEQ ID NO: 6.
  • the IL-2 mutein comprises a mutation at positions 73 and 76; 73 and 100; 73 and 138; 76 and 100; 76 and 138; 100 and 138; 73, 76, and 100; 73, 76, and 138; 73, 100, and 138; 76, 100 and 138; or at each of 73, 76, 100, and 138 that correspond to SEQ ID NO: 15.
  • the IL-2 mutein comprises a mutation at positions 53 and 56; 53 and 80; 53 and 118; 56 and 80; 56 and 118; 80 and 1 18; 53, 56, and 80; 53, 56, and 118; 53, 80, and 118; 56, 80 and 118; or at each of 53, 56, 80, and 118 that correspond to SEQ ID NO: 6.
  • the term corresponds to as reference to a SEQ ID NOs: 6 or 15 refer to how the sequences would align with default settings for alignment software, such as can be used with the NCBI website.
  • the mutation is leucine to isoleucine.
  • the IL-2 mutein can comprise one more isoleucines at positions 73, 76, 100, or 138 that correspond to SEQ ID NO:
  • the mutein comprises a mutation at L53 that correspond to SEQ ID NO:
  • the mutein comprises a mutation at L56 that correspond to SEQ ID NO: 6. In some embodiments, the mutein comprises a mutation at L80 that correspond to SEQ ID NO: 6. In some embodiments, the mutein comprises a mutation at Ll 18 that correspond to SEQ ID NO: 6. In some embodiments, the mutation is leucine to isoleucine. In some
  • the mutein also comprises a mutation as position 69, 74, 88, 125, or any combination thereof in these muteins that correspond to SEQ ID NO: 6.
  • the mutation is a V69A mutation.
  • the mutation is a Q74P mutation.
  • the mutation is a N88D or N88R mutation.
  • the mutation is a C125A or C125S mutation.
  • the IL-2 mutein comprises a mutation at one or more of positions 49, 51, 55, 57, 68, 89, 91, 94, 108, and 145 that correspond to SEQ ID NO: 15 or one or more positions 29, 31, 35, 37, 48, 69, 71, 74, 88, and 125 that correspond to SEQ ID NO: 6.
  • the substitutions can be used alone or in combination with one another.
  • the IL-2 mutein comprises substitutions at 2, 3, 4, 5, 6, 7, 8, 9, or each of positions 49, 51, 55, 57, 68, 89, 91, 94, 108, and 145.
  • Non-limiting examples such combinations include, but are not limited to, a mutation at positions 49, 51, 55, 57, 68, 89, 91, 94, 108, and 145; 49, 51, 55, 57, 68, 89, 91, 94, and 108; 49, 51, 55, 57, 68, 89, 91, and 94; 49, 51, 55, 57, 68, 89, and 91; 49, 51, 55, 57, 68, and 89; 49, 51, 55, 57, and 68; 49, 51, 55, and 57; 49, 51, and 55; 49 and 51; 51, 55, 57, 68, 89, 91, 94, 108, and 145; 51, 55, 57, 68, 89, 91, 94, and 108; 51, 55, 57, 68, 89, 91, and 94; 51, 55, 57, 68, 89, 91, and 94; 51,
  • the IL-2 mutein comprises a mutation at one or more positions of 35, 36, 42, 104, 115, or 146 that correspond to SEQ ID NO: 15 or the equivalent positions at SEQ ID NO: 6 ( e.g . positions 15, 16, 22, 84, 95, or 126).
  • These mutations can be combined with the other leucine to isoleucine mutations described herein or the mutation at positions 73, 76,
  • the mutation is a E35Q, H36N, Q42E, D104N, El 15Q, or Q146E, or any combination thereof.
  • one or more of these substitutions is wildtype.
  • the mutein comprises a wild-type residue at one or more of positions 35, 36, 42, 104, 115, or 146 that correspond to SEQ ID NO: 15 or the equivalent positions at SEQ ID NO: 6 (e.g. positions 15, 16, 22, 84, 95, and 126).
  • the IL-2 mutein comprises a N49S mutation that corresponds to SEQ ID NO: 15. In some embodiments, the IL-2 mutein comprises a Y51S or a Y51H mutation that corresponds to SEQ ID NO: 15. In some embodiments, the IL-2 mutein comprises a K55R mutation that corresponds to SEQ ID NO: 15.
  • the IL-2 mutein comprises a T57A mutation that corresponds to SEQ ID NO: 15. In some embodiments, the IL-2 mutein comprises a K68E mutation that corresponds to SEQ ID NO: 15. In some embodiments, the IL-2 mutein comprises a V89A mutation that corresponds to SEQ ID NO: 15. In some embodiments, the IL-2 mutein comprises a N91R mutation that corresponds to SEQ ID NO: 15. In some embodiments, the IL-2 mutein comprises a Q94P mutation that corresponds to SEQ ID NO: 15. In some embodiments, the IL-2 mutein comprises a N108D or a N108R mutation that corresponds to SEQ ID NO: 15.
  • the IL-2 mutein comprises a C145A or C145S mutation that corresponds to SEQ ID NO: 15. These substitutions can be used alone or in combination with one another. In some embodiments, the mutein comprises each of these substitutions. In some embodiments, the mutein comprises 1, 2, 3, 4, 5, 6, 7, or 8 of these mutations. In some embodiments, the mutein comprises a wild-type residue at one or more of positions 35, 36, 42, 104, 115, or 146 that correspond to SEQ ID NO: 15 or the equivalent positions at SEQ ID NO: 6 (e.g. positions 15, 16, 22, 84, 95, and 126).
  • the IL-2 mutein comprises a N29S mutation that corresponds to SEQ ID NO: 6. In some embodiments, the IL-2 mutein comprises a Y31 S or a Y31H mutation that corresponds to SEQ ID NO: 6. In some embodiments, the IL-2 mutein comprises a K35R mutation that corresponds to SEQ ID NO: 6. In some embodiments, the IL-2 mutein comprises a T37A mutation that corresponds to SEQ ID NO: 6. In some embodiments, the IL-2 mutein comprises a K48E mutation that corresponds to SEQ ID NO: 6. In some embodiments, the IL-2 mutein comprises a V69A mutation that corresponds to SEQ ID NO: 6. In some embodiments, the IL-2 mutein comprises a N71R mutation that corresponds to SEQ ID NO: 6. In some embodiments, the IL-2 mutein comprises a Q74P mutation that corresponds to SEQ ID NO: 6.
  • the IL-2 mutein comprises a N88D or a N88R mutation that corresponds to SEQ ID NO: 6. In some embodiments, the IL-2 mutein comprises a C125A or C125S mutation that corresponds to SEQ ID NO: 6. These substitutions can be used alone or in combination with one another. In some embodiments, the mutein comprises 1, 2, 3, 4, 5, 6, 7, or 8 of these mutations. In some embodiments, the mutein comprises each of these substitutions.
  • the mutein comprises a wild-type residue at one or more of positions 35, 36, 42, 104, 115, or 146 that correspond to SEQ ID NO: 15 or the equivalent positions at SEQ ID NO: 6 (e.g. positions 15, 16, 22, 84, 95, and 126).
  • one or more of positions 35, 36, 42, 104, 115, or 146 that correspond to SEQ ID NO: 15 or the equivalent positions at SEQ ID NO: 6 are wild-type (e.g.are as shown in SEQ ID NOs: 6 or 15).
  • 2, 3, 4, 5, 6, or each of positions 35, 36, 42, 104, 115, or 146 that correspond to SEQ ID NO: 15 or the equivalent positions at SEQ ID NO: 6 are wild-type.
  • the IL-2 mutein comprises a sequence of:
  • the IL-2 mutein comprises a sequence of:
  • the IL-2 mutein comprises a sequence of:
  • the IL-2 mutein comprises a sequence of:
  • the IL-2 mutein sequences described herein do not comprise the IL-2 leader sequence.
  • the IL-2 leader sequence can be represented by the sequence of
  • the sequences illustrated above can also encompass peptides without the leader sequence.
  • SEQ ID NOs; 16-20 are illustrated with only mutation at one of postions 73, 76, 100, or 138 that correspond to SEQ ID NO: 15 or positions at one or more of positions 53, 56, 80, or 118 that correspond to SEQ ID NO: 6, the peptides can comprises one, two, three or 4 of the mutations at these positions.
  • the substitution at each position is isoleucine or other type of conservative amino acid substitution.
  • the leucine at the recited positions are substituted with, independently, isoleucine, valine, methionine, or phenylalanine.
  • the IL-2 mutein molecule is fused to a Fc Region or other linker region as described herein. Examples of such fusion proteins can be found in US9580486, US7105653, US9616105, US 9428567, US2017/0051029, WO2016/164937,
  • the Fc Region comprises what is known as the LALA mutation. Using the Rabat numbering of the Fc region, this would correspond to L247A, L248A, and G250A. In some embodiments, using the EU numbering of the Fc region, the Fc region comprises a L234A mutation, a L235A mutation, and/or a G237A mutatoin mutation.
  • the Fc portion can comprise mutations that correspond to these residues.
  • the Fc Region comprises N297G or N297A (kabat numbering) mutations.
  • the Rabat numbering is based upon a full- length sequence, but would be used in a fragment based upon a traditional alignment used by one of skill in the art for the Fc region.
  • the Fc Region comprises a sequence of:
  • DRTHTCPPCPAPEAAGAPSVFLFPPRPRDTLMISRTPEVTCV VVD V SHEDPEVRFNW YVDGVEVHNARTRPREEQYNSTYR VV S VLTVLHQDWLNGREYRCR V SNRALP APIERTISRARG QPREPQ V YTLPP SREEMTRN Q VSLT CL VRGF YP SDI A VEWE SNGQPENNYRTTPPVLDSDGSFFLYSRLTVDRSRWQQGNV FSCSVMHEALHNHYTQRSLSLSPG. (SEQ ID NO: 21)
  • the IL-2 mutein is linked to the Fc Region.
  • linkers are gly cine/ serine linkers.
  • a glycine/serine linkers can be a sequence of GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 22) or GGGGSGGGGSGGGGS (SEQ ID NO: 30). This is simply a non-limiting example and the linker can have varying number of GGGGS (SEQ ID NO: 23) or GGGGA repeats (SEQ ID NO: 29).
  • the linker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the GGGGS (SEQ ID NO: 23) or GGGGA repeats (SEQ ID NO: 29) repeats.
  • the IL-2/Fc Fusion can be represented by the formula of Zi L-2 M-E gs -Z Fc , wherein ZIL-2M is a IL-2 mutein as described herein, L gs is a linker sequence as described herein ( e.g . glycine/serine linker) and Z Fc is a Fc region described herein or known to one of skill in the art.
  • the formula can be in the reverse orientation Z Fc -L gs -Zi L- 2 M ⁇
  • the IL-2/Fc fusion comprises a sequence of
  • the IL-2/Fc Fusion comprises a sequence selected from the following table, Table 2:
  • the table also provides sequences for a variety of Fc domains or variants that the IL-2 can be fused with:
  • sequences shown in the table or throughout comprise or don’t comprise one or more mutations that correspond to positions L53, L56, L80, and Ll 18. .
  • sequences shown in the table or throughout the present application comprise or don’t comprise one or more mutations that correspond to positions L59I, L63I, I24L, L94I, L96I or L 1321 or other substitutions at the same positions.
  • the mutation is leucine to isoleucine.
  • the mutein does not comprise another mutation other than as shown or described herein.
  • the peptide comprises a sequence of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO:
  • SEQ ID NO: 46 SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, or SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, or SEQ ID NO: 60.
  • the protein comprises a IL-2 mutein as provided for herein.
  • a polypeptide is provided comprising SEQ ID NO: 59 or SEQ ID NO: 60, wherein at least one of Xi, X 2 , X 3 , and X 4 is I and the remainder are L or I.
  • Xi, X 2 , and X 3 are L and X 4 is I. In some embodiments, Xi, X 2 , and X 4 are L and X 3 is I. In some embodiments, X 2 , X 3 , and X 4 are L and Xi is I. In some embodiments, Xi, X 3 , and X 4 are L and X 2 is I. In some embodiments, X 3 and X 2 are L and X 3 and X 4 are I. In some embodiments, Xi and X 3 are L and X 2 and X are I. In some embodiments, Xi and X 3 are L and X 2 and X are I. In some embodiments, Xi and X are L and X 2 and X 3 are I.
  • X 2 and X 3 are L and Xi and X 4 are I. In some embodiments, X 2 and X 4 are L and Xi and X 3 are I. In some embodiments, X 3 and X 4 are L and Xi and X 2 are I. In some embodiments, Xi, X 2 , and X 3 are L and X 4 is I. In some embodiments, X 2 , X 3 , and X are L and Xi is I. In some embodiments, Xi, X 3 , and X are L and X 2 is I. In some embodiments, Xi, X 2 , and X 4 are L and X 3 is I.
  • the Fc portion of the fusion is not included.
  • the peptide consists essentially of a IL-2 mutein provided for herein.
  • the protein is free of a Fc portion.
  • the compound comprises an amino acid sequence of SEQ ID NO: 53, 54,
  • the compound comprises an amino acid sequence of SEQ ID NO: 53, 54, 55, or 56 with or without a C125A or C125S mutation.
  • the residue at position 125 is C, S, or A.
  • the compound comprises an amino acid sequence of SEQ ID NO: 59 or SEQ ID NO: 60, wherein at least one of Xi, X 2 , X 3 , and X 4 is I and the remainder are L or I.
  • the protein comprises a IL-2 mutein as provided for herein.
  • a polypeptide comprising SEQ ID NO: 59 or SEQ ID NO: 60, wherein at least one of Xi, X 2 , X 3 , and X 4 is I and the remainder are L or I.
  • Xi, X 2 , and X 3 are L and X is I.
  • Xi, X 2 , and X 4 are L and X 3 is I.
  • X 2 , X 3 , and X 4 are L and Xi is I.
  • Xi, X 3 , and X 4 are L and X 2 is I.
  • Xi and X 2 are L and X 3 and X 4 are I.
  • Xi and X 3 are L and X 2 and X 4 are I.
  • Xi and X 3 are L and X 2 and X 4 are I.
  • Xi and X 3 are L and X 2 and X 4 are I.
  • Xi and X 3 are L and X
  • Xi and X are L and X 2 and X 3 are I. In some embodiments, X 2 and X 3 are L and Xi and X 4 are I. In some embodiments, X 2 and X 4 are L and Xi and X 3 are I. In some embodiments, X 3 and X 4 are L and Xi and X 2 are I. In some embodiments, Xi, X 2 , and X 3 are L and X 4 is I. In some embodiments, X 2 , X 3 , and X 4 are L and Xi is I. In some embodiments, Xi, X 3 , and X are L and X 2 is I. In some embodiments, Xi, X 2 , and X are L and X 3 is I. In some embodiments, Xi, X 2 , and X are L and X 3 is I.
  • Each of the proteins may also be considered to have the C125S and the LALA and/or G237A mutations as provided for herein.
  • the C125 substitution can also be C125A as described throughout the present application.
  • an IL-2 mutein molecule comprises at least 60, 70, 80, 85, 90, 95, or 97% sequence identity or homology with a naturally occurring human IL-2 molecule, e.g., a naturally occurring IL-2 sequence disclosed herein or those that incorporated by reference.
  • the IL-2 muteins can be part of a bi-specific molecule with a tethering moiety, such as a MAdCAM antibody that will target the IL-2 mutein to a MAdCAM expressing cell.
  • the bispecific molecule can be produced from two polypeptide chains. In some embodiments, the following can be used:
  • the proteins can be produced with or without a Cl 25 A or Cl 25 S mutation in the IL-2 mutein.
  • IL-2 muteins that can be included are illustrated herein, such as, but not limited to, a sequence of SEQ ID NO: 59 or SEQ ID NO: 60..
  • the constant kappa domain in any of the light chains can be replaced with a constant lambda domain.
  • GITR(CD357) is a cell surface marker present on Tregs. Blockade of the GITR-GITRL interaction maintains Treg function.
  • a therapeutic compound comprises an IIC binding entity that binds GITR-expressing Treg cells and a targeting moiety that targets the therapeutic compound to the target tissue of interest.
  • a therapeutic compound comprises an anti-GITR antibody molecule, e.g., anti-GITR antibody molecule that inhibit binding of GITR to GITRL.
  • a therapeutic compound comprises an anti-GITR antibody molecule, anti-GITR antibody molecule that inhibit binding of GITR to GITRL, and PD-l agonist, IL-2 mutein molecule, or other effector described herein.
  • the therapeutic compound that comprises a GITR binder effects accumulation of GITR-expressing Tregs at the site targeted by the targeting moiety of the therapeutic compound, e.g., a transplant or site of organ injury.
  • Effector binding/modulating moiety can comprise an agonistic BTNL2 molecule. While not wishing to be bound by theory it is believed that agonistic BTNL2 molecules induce Treg cells.
  • An agonistic BTNL2 molecule as that term as used herein, refers to a polypeptide having sufficient BTNL2 sequence that, as part of a therapeutic compound, it induces Treg cells.
  • a BTNL2 molecule has at least 60, 70, 80, 90, 95, 99, or 100% sequence identity, or substantial sequence identity, with a naturally occurring butyrophilin.
  • an effector binding/modulating moiety an antagonistic BTNL8 molecule.
  • a therapeutic compound can comprise an effector binding/modulating moiety that promotes an immuno-suppressive local microenvironment, e.g., by providing in the proximity of the target, a substance that inhibits or minimizes attack by the immune system of the target, referred to herein a SM binding/modulating moiety.
  • the SM binding/modulating moiety comprises a molecule that inhibits or minimizes attack by the immune system of the target (referred to herein as an SM binding/modulating moiety).
  • a therapeutic compound comprises an SM binding/modulating moiety that binds and accumulates a soluble substance, e.g., an endogenous or exogenous substance having immunosuppressive function.
  • a therapeutic compound comprises an SM binding/modulating moiety, e.g., a CD39 molecule or a CD73 molecule or alkaline phosphatase molecule, that binds, inhibits, sequesters, degrades or otherwise neutralizes a soluble substance, typically and endogenous soluble substance, e.g., ATP in the case of a CD39 molecule or alkaline phosphatase molecule, or AMP in the case of a CD73 molecule, that promotes immune attack.
  • a therapeutic compound comprises an SM binding/modulating moiety that comprises an immune-suppressive substance, e.g. a fragment of protein that is immunosuppressive.
  • a therapeutic compound can comprise an ICSM binding/modulating moiety that inhibits or antagonizes a stimulatory, e.g., co-stimulatory binding pair, e.g., 0X40 and OX40L.
  • the ICSM binding/modulating moiety can bind and antagonize either member of the pair.
  • the ICSM binding/modulating moiety comprises an antibody molecule that binds and antagonizes either member of a stimulatory, e.g., co-stimulatory binding pair.
  • the ICSM binding/modulating moiety comprises antagonistic analog of one of the members of the binding pair.
  • the ICSM binding/modulating moiety can comprise a soluble fragment of one of the members that binds the other.
  • the analog will have at least 50, 60, 70, 80, 90, 95, or 98% homology or sequence identity with a naturally occurring member that binds the target member of the pair.
  • the ICSM binding/modulating moiety typically binds but does not activate, or allow endogenous counter member to bind and activate.
  • an ICSM binding/modulating member can comprise any of the following:
  • an antibody molecule that binds the 0X40 immune cell member and antagonizes stimulation e.g., by blocking binding of endogenous OX40L counter member
  • an antibody molecule that binds OX40L counter member and antagonizes stimulation e.g., by blocking effective binding of the endogenous OX40L counter member to the 0X40 immune cell member
  • the ICSM binding/modulating moiety e.g., an antibody molecule or an antagonistic analog or of the counter member, can bind to CD2, ICOS, CD40L, CD28, LFA1, SLAM, TIM1, CD30, 0X40 (CD134), 41BB (CD137), CD27, HVEM, DR3, GITR, BAFFR, TACI, BCMA, or CD30, CD40.
  • the ICSM binding/modulating moiety e.g., an antibody molecule or an antagonistic analog or of the counter member, can bind to B7.1, B7.2, ICOSL (B7-H2, B7RP1), LFA3, CD48, CD58, ICAM1, SLAM, TIM4, CD40, CD30L, OX40L (CD252), 41BBL (CD137L), CD70, LIGHT, TL1A, GITRL, BAFF, APRIL, or CD30, CD40L.
  • B7.1, B7.2, ICOSL B7-H2, B7RP1
  • CD40 CD30L
  • OX40L CD252
  • 41BBL CD137L
  • CD70 LIGHT
  • TL1A TL1A
  • GITRL BAFF
  • BAFF APRIL
  • CD30 CD40L
  • the ICSM binding/modulating molecule binds, and antagonizes, an activating or costimulatory molecule, e.g., a costimulatory molecule, present on an immune cell, or binds the counter member preventing the counter member from activating the
  • the ICSM comprises an antagonistic antibody molecule e.g., an antibody molecule that binds the costimulatory molecule on an immune cell or binds the counter member of the ICSM, preventing the counter member from activating the costimulatory molecule on the immune cell, and results in inhibiting the activity of the costimulatory molecule.
  • the ICSM comprises an antagonistic counterpart molecule, e.g., a fragment of a molecule that binds the costimulatory molecule, and results in the inhibition of the costimulatory molecule activity.
  • one member of the binding pair will be on the surface of an immune cell, e.g., a T, B, or NK cell or dendritic cell, while the counter member will be on another immune cell, or an APC such as a dendritic cell_ or on non-immune cells such as smooth cells, or endothelial cells.
  • an immune cell e.g., a T, B, or NK cell or dendritic cell
  • the counter member will be on another immune cell, or an APC such as a dendritic cell_ or on non-immune cells such as smooth cells, or endothelial cells.
  • DONOR TISSUE Therapeutic compounds and methods described herein can be used in conjunction with a transplantation of donor tissue into a subject and can minimizes rejection of, minimizes immune effector cell mediated damage to, prolongs acceptance of, or prolongs the functional life of, donor transplant tissue.
  • the tissue can be xenograft or allograft tissue.
  • Transplanted tissue can comprise all or part of an organ, e.g., a liver, kidney, heart, pancreas, thymus, skin or lung.
  • therapeutic compounds described herein reduce, or eliminate the need for systemic immune suppression.
  • Therapeutic compounds and methods described herein can also be used to treat GVHD.
  • host cells are coated with a therapeutic compound that comprises, as an effector binding/modulating moiety, a PD-L1 molecule.
  • a target molecule is the target to which a targeting moiety binds.
  • a targeting moiety is selected that binds a product of an allele present on donor tissue and which is not expressed by the subject (recipient) or at expressed at a different level (e.g. reduced or substantially reduced).
  • Therapeutic compounds and methods described herein can be used to treat a subject having or at risk for having an unwanted autoimmune response, e.g., an auto immune response in Type 1 Diabetes, Multiple Sclerosis, Cardiomyositis, vitiligo, alopecia, inflammatory bowel disease (IBD, e.g. Crohn’s disease or ulcerative colitis), Sjogren’s syndrome, focal segmented glomerular sclerosis (FSGS), scleroderma/systemic sclerosis (SSc) or rheumatoid arthritis.
  • the treatment minimizes rejection of, minimizes immune effector cell mediated damage to, prolongs the survival of subject tissue undergoing, or a risk for, autoimmune attack.
  • Table 3 provides target molecules for several autoimmune indications and organ/cell types.
  • a target molecule is the target to which a targeting moiety binds.
  • autoimmune disorders and diseases that can be treated with the compounds described herein include, but are not limited to, Myocarditis, Postmyocardial infarction syndrome, Postpericardiotomy syndrome, Subacute bacterial endocarditis, Anti-
  • Glomerular Basement Membrane nephritis Interstitial cystitis, Lupus nephritis, membranous glomerulonephropathy, Chronic Kidney Disease (“CKD”), Autoimmune hepatitis, Primary biliary cirrhosis, Primary sclerosing cholangitis, Anti synthetase syndrome, alopecia areata, autoimmune angioedema, autoimmune progesterone dermatitis, autoimmune urticaria, bullous pemphigoid, cicatricial pemphigoid, dermatitis herpetiformis, discoid lupus erythematosus, epidermolysis bullosa acquisita, erythema nodosum, gestational pemphigoid, hidradenitis suppurativa, lichen planus, lichen sclerosus, linear iga disease (lad), morphea, pemphigus vulgaris,
  • Enthesitis-related arthritis Eosinophilic fasciitis, Felty syndrome, IgG4-related disease, Juvenile, Arthritis, Lyme disease (Chronic), Mixed connective tissue disease (MCTD), Palindromic rheumatism, Parry Romberg syndrome, Parsonage-Turner syndrome, Psoriatic arthritis, Reactive arthritis, Relapsing polychondritis, Retroperitoneal fibrosis, Rheumatic fever, Rheumatoid arthritis, Sarcoidosis, Schnitzler syndrome, Systemic Lupus Erythematosus (SLE),
  • Undifferentiated connective tissue disease UCTD
  • Dermatomyositis Fibromyalgia
  • Inclusion body myositis Myositis, Myasthenia gravis
  • Neuromyotonia Paraneoplastic cerebellar degeneration
  • Polymyositis Acute disseminated encephalomyelitis (ADEM)
  • Acute motor axonal neuropathy Acute anti-N-Methyl-D-Aspartate (anti-NMDA) Receptor Encephalitis, Balo concentric sclerosis, Bickerstaff s encephalitis, Chronic inflammatory demyelinating
  • Streptococcus Progressive inflammatory neuropathy, Restless leg syndrome, Stiff person syndrome, Sydenham chorea, Transverse myelitis, Autoimmune retinopathy,
  • AIED Autoimmune inner ear disease
  • Meniere's disease Behcet’s disease
  • Eosinophilic granulomatosis with polyangiitis EGPA
  • Giant cell arteritis GAA
  • Granulmatosis with polyangiitis GAA
  • IgA vasculitis IgAY
  • Kawasaki’s disease Leukocytoclastic vasculitis, Lupus vasculitis, Rheumatoid vasculitis, Microscopic polyangiitis (MPA), Polyarteritis nodosa (PAN), Polymyalgia rheumaticia, Vasculitis, Primary Immune Deficiency, and the like.
  • MPA Polyarteritis nodosa
  • PAN Polymyalgia rheumaticia
  • Vasculitis Primary Immune Deficiency, and the like.
  • autoimmune disorders and diseases include, but are not limited to, Chronic fatigue syndrome, Complex regional pain syndrome, Eosinophilic esophagitis, Gastirtis, Interstitial lung disease, POEMS syndrome, Raynaud’s phenomenon, Primary immunodeficiency, Pyoderma gangrenosum, Agammaglobulinemia, Anyloidosis, Anyotrophic lateral sclerosis, Anti-tubular basement membrane nephritis, Atopic allergy, Atopic dermatitis, Autoimmune peripheral neuropathy, Blau syndrome, Castleman’s disease, Chagas disease, Chronic obstructive pulmonary disease, Chronic recurrent multifocal osteomyelitis, Complement component 2 deficiency, Contact dermatitis, Cushing’s syndrome, Cutaneous leukocytoclastic angiitis, Dego’ deiase, Eczema, Eosinophilic gastroente
  • the autoimmune disorder does not comprise pemphigus
  • the autoimmune disorder does not comprise pemphigus foliaceus. In some embodiments, the autoimmune disorder does not comprise bullous pemphigoid. In some embodiments, the autoimmune disorder does not comprise Goodpasture’s Disease. In some embodiments, the autoimmune disorder does not comprise psoriasis. In some embodiments, the autoimmune disorder does not comprise a skin disorder. In some embodiments, the disorder does not comprise a neoplastic disorder, e.g., cancer.
  • a therapeutic compound comprises a specific targeting moiety functionally associated with an effector binding/modulating moiety.
  • the specific targeting moiety and effector binding/modulating moiety are linked to one another by a covalent or noncovalent bond, e.g., a covalent or non-covalent bond directly linking the one to the other.
  • a specific targeting moiety and effector binding/modulating moiety are linked, e.g., covalently or noncovalently, through a linker moiety.
  • a polypeptide sequence comprising the specific targeting moiety and a polypeptide sequence can be directly linked to one another or linked through one or more linker sequences.
  • the linker moiety comprises a polypeptide.
  • Linkers are not, however, limited to polypeptides.
  • a linker moiety comprises other backbones, e.g., a non-peptide polymer, e.g., a PEG polymer.
  • a linker moiety can comprise a particle, e.g., a nanoparticle, e.g., a polymeric nanoparticle.
  • a linker moiety can comprise a branched molecule, or a dendrimer.
  • the effector binding/modulating moiety comprises an ICIM binding/modulating moiety (which binds an effector like PD-l) structures that result in clustering in the absence of target binding should be avoided as they may cause clustering in the absence of target binding.
  • the therapeutic compound has a structure, e.g., the copies of an ICIM are sufficiently limited, such that clustering in the absence of target binding is minimized or substantially eliminated, or eliminated, or is sufficiently minimized that substantial systemic immune suppression does not occur.
  • a therapeutic compound comprises a polypeptide comprising a specific targeting moiety covalently or non-covalently conjugated to an effector
  • a therapeutic molecule comprises a fusion protein having comprising a specific targeting moiety fused, e.g., directly or through a linking moiety comprising one or more amino acid residues, to an effector binding/modulating moiety.
  • a therapeutic molecule comprises a polypeptide comprising a specific targeting moiety linked by a non-covalent bond or a covalent bond, e.g., a covalent bond other than a peptide bond, e.g., a sulfhydryl bond, to an effector binding/modulating moiety.
  • a therapeutic compound comprises polypeptide, e.g., a fusion polypeptide, comprising:
  • a specific targeting moiety comprising a target ligand binding molecule
  • l.c a specific targeting moiety comprising an antibody molecule
  • l.d a specific targeting moiety comprising a single chain antibody molecule, e.g., a scFv domain
  • a specific targeting moiety comprising a first of the light or heavy chain variable region of an antibody molecule, and wherein the other variable region is covalently or non covalently associated with the first;
  • an effector binding/modulating moiety comprising an effector specific binding polypeptide
  • an effector binding/modulating moiety comprising an effector ligand binding molecule
  • an effector binding/modulating moiety comprising a single chain antibody molecule, e.g., a scFv domain; or
  • an effector binding/modulating moiety comprising a first of the light or heavy chain variable region of an antibody molecule, and wherein the other variable region is covalently or non covalently associated with the first.
  • a therapeutic compound comprises l.a and 2. a.
  • a therapeutic compound comprises l.a and 2.b.
  • a therapeutic compound comprises l.a and 2.c.
  • a therapeutic compound comprises l.a and 2.d.
  • a therapeutic compound comprises l.a and 2.e.
  • a therapeutic compound comprises l.b and 2. a.
  • a therapeutic compound comprises l.b and 2.b.
  • a therapeutic compound comprises l.b and 2.c.
  • a therapeutic compound comprises l.b and 2.d.
  • a therapeutic compound comprises l.b and 2.e.
  • a therapeutic compound comprises l.c and 2. a.
  • a therapeutic compound comprises l.c and 2.b.
  • a therapeutic compound comprises l.c and 2.c.
  • a therapeutic compound comprises l.c and 2.d.
  • a therapeutic compound comprises l.c and 2.e. In some embodiments, a therapeutic compound comprises l.d and 2. a.
  • a therapeutic compound comprises l.d and 2.b.
  • a therapeutic compound comprises l.d and 2.c.
  • a therapeutic compound comprises l.d and 2.d.
  • a therapeutic compound comprises l.d and 2.e.
  • a therapeutic compound comprises l.e and 2. a.
  • a therapeutic compound comprises l.e and 2.b.
  • a therapeutic compound comprises l.e and 2.c.
  • a therapeutic compound comprises l.e and 2.d.
  • a therapeutic compound comprises l.e and 2.e.
  • Therapeutic compounds disclosed herein can, for example, comprise a plurality of effector binding/modulating and specific targeting moieties. Any suitable linker or platform can be used to present the plurality of moieties. The linker is typically coupled or fused to one or more effector binding/modulating and targeting moieties.
  • two (or more) linkers associate, either covalently or
  • the linker can comprise an Fc region and two Fc regions associate with one another.
  • the linker regions can self associate, e.g., as two identical Fc regions.
  • the linker regions are not capable of, or not capable of substantial, self association, e.g., the two Fc regions can be members of a knob and hole pair.
  • Non-limiting exemplary configurations of therapeutic compounds comprise the following (e.g., in N to C terminal order):
  • Rl, R2, R3, and R4 each independently comprises an effector binding/modulating moiety, e.g., an ICIM binding/modulating moiety, an IIC binding/modulating moiety, ICSM binding/modulating moiety, or an SM binding/modulating moiety; a specific targeting moiety; or is absent;
  • Linker Region A and Linker B comprise moieties that can associate with one another, e.g., Linker A and Linker B each comprises an Fc moiety provided that an effector
  • binding/modulating moiety and a specific targeting moiety are present.
  • Rl comprises an effector binding/modulating moiety, e.g., an ICIM binding/modulating moiety, an IIC binding/modulating moiety, ICSM binding/modulating moiety, or an SM binding/modulating moiety, or is absent;
  • an effector binding/modulating moiety e.g., an ICIM binding/modulating moiety, an IIC binding/modulating moiety, ICSM binding/modulating moiety, or an SM binding/modulating moiety, or is absent;
  • R2 comprises a specific targeting moiety, or is absent
  • R3 comprises an effector binding/modulating moiety, e.g., an ICIM binding/modulating moiety, an IIC binding/modulating moiety, ICSM binding/modulating moiety, or an SM binding/modulating moiety, or is absent;
  • an effector binding/modulating moiety e.g., an ICIM binding/modulating moiety, an IIC binding/modulating moiety, ICSM binding/modulating moiety, or an SM binding/modulating moiety, or is absent;
  • R4 comprises a specific targeting moiety, or is absent
  • Linker Region A and Linker B comprise moieties that can associate with one another, e.g., Linker A and Linker B each comprises an Fc moiety, provided that one of Rl or R3 is present and one of R2 or R4 is present.
  • Rl comprises a specific targeting moiety, or is absent
  • R2 comprises an effector binding/modulating moiety, e.g., an ICIM binding/modulating moiety, an IIC binding/modulating moiety, ICSM binding/modulating moiety, or an SM binding/modulating moiety, or is absent;
  • an effector binding/modulating moiety e.g., an ICIM binding/modulating moiety, an IIC binding/modulating moiety, ICSM binding/modulating moiety, or an SM binding/modulating moiety, or is absent;
  • R3 comprises a specific targeting moiety, or is absent
  • R4 comprises an effector binding/modulating moiety, e.g., an ICIM binding/modulating moiety, an IIC binding/modulating moiety, ICSM binding/modulating moiety, or an SM binding/modulating moiety, or is absent;
  • an effector binding/modulating moiety e.g., an ICIM binding/modulating moiety, an IIC binding/modulating moiety, ICSM binding/modulating moiety, or an SM binding/modulating moiety, or is absent;
  • Linker Region A and Linker B comprise moieties that can associate with one another, e.g., Linker A and Linker B each comprises an Fc moiety, provided that one of Rl or R3 is present and one of R2 or R4 is present.
  • Non-limiting examples include, but are not limited to:
  • Rl, R2, R3 and R4 each independently comprise: an effector binding modulating moiety that activates an inhibitory receptor on an immune cell, e.g., a T cell or a B cell, e.g., a PD-L1 molecule or a functional anti-PD-l antibody molecule (an agonist of PD-l); a specific targeting moiety; or is absent;
  • an effector binding modulating moiety that activates an inhibitory receptor on an immune cell, e.g., a T cell or a B cell, e.g., a PD-L1 molecule or a functional anti-PD-l antibody molecule (an agonist of PD-l); a specific targeting moiety; or is absent;
  • an effector binding moiety and a specific targeting moiety are present.
  • Linker A and Linker B comprise Fc moieties (e.g., self pairing Fc moieties).
  • Rl and R3 independently comprise an effector binding modulating moiety that activates an inhibitory receptor on an immune cell, e.g., a T cell or a B cell, e.g., a PD-L1 molecule or an functional anti-PD-l antibody molecule (an agonist of PD-l); and
  • R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.
  • Linker A and Linker B comprise Fc moieties (e.g., self pairing Fc moieties).
  • Rl and R3 independently comprise a functional anti-PD-l antibody molecule (an agonist of PD- 1);
  • R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.
  • Linker A and Linker B comprise Fc moieties (e.g., self pairing Fc moieties).
  • Rl and R3 independently comprise specific targeting moieties, e.g., an anti-tissue antigen antibody
  • R2 and R4 independently comprise a functional anti-PD-l antibody molecule (an agonist of PD- 1), e.g., an scFv molecule.
  • Linker A and Linker B comprise Fc moieties (e.g., self pairing Fc moieties).
  • Rl and R3 independently comprise a PD-Ll molecule (an agonist of PD-l);
  • R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen; and
  • Linker A and Linker B comprise Fc moieties (e.g., self pairing Fc moieties).
  • Rl and R3 independently comprise specific targeting moieties, e.g., an anti- tissue antigen antibody
  • R2 and R4 independently comprise a PD-L1 molecule (an agonist of PD-l).
  • Linker A and Linker B comprise Fc moieties (e.g., self pairing Fc moieties).
  • Rl, R2, R3 and R4 each independently comprise: an SM binding/modulating moiety which modulates, e.g., binds and inhibits, sequesters, degrades or otherwise neutralizes a substance, e.g., a soluble molecule that modulates an immune response, e.g., ATP or AMP, e.g., a CD39 molecule or a CD73 molecule; a specific targeting moiety; or is absent;
  • an SM binding/modulating moiety which modulates, e.g., binds and inhibits, sequesters, degrades or otherwise neutralizes a substance, e.g., a soluble molecule that modulates an immune response, e.g., ATP or AMP, e.g., a CD39 molecule or a CD73 molecule; a specific targeting moiety; or is absent;
  • Linker A and Linker B comprise Fc moieties (e.g., self pairing Fc moieties or Fc moieties that do not, or do not substantially self pair).
  • Rl and R3 independently comprise an SM binding/modulating moiety which modulates, e.g., binds and inhibits, sequesters, degrades or otherwise neutralizes a substance, e.g., a soluble molecule that modulates an immune response, e.g., ATP or AMP, e.g., a CD39 molecule or a CD73 molecule; and
  • R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.
  • Linker A and Linker B comprise Fc moieties (e.g., self pairing Fc moieties or Fc moieties that do not, or do not substantially self pair).
  • Rl and R3 independently comprise a CD39 molecule or a CD73 molecule
  • R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.
  • Linker A and Linker B comprise Fc moieties (e.g., self pairing Fc moieties or Fc moieties that do not, or do not substantially self pair).
  • Fc moieties e.g., self pairing Fc moieties or Fc moieties that do not, or do not substantially self pair.
  • Rl and R3 each comprises a CD39 molecule
  • R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen
  • Linker A and Linker B comprise Fc moieties (e.g., self pairing Fc moieties or Fc moieties that do not, or do not substantially self pair).
  • Rl and R3 each comprises a CD73 molecule
  • R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.
  • Linker A and Linker B comprise Fc moieties (e.g., self pairing Fc moieties or Fc moieties that do not, or do not substantially self pair).
  • Rl and R3 comprises a CD39 molecule and the other comprises a CD73 molecule; and R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.
  • Linker A and Linker B comprise Fc moieties (e.g., self pairing Fc moieties or Fc moieties that do not, or do not substantially self pair).
  • Rl, R2, R3 and R4 each independently comprise: an HLA-G molecule; a specific targeting moiety; or is absent;
  • HLA-G molecule and a specific targeting moiety are present.
  • Linker A and Linker B comprise Fc moieties (e.g., self pairing Fc moieties or Fc moieties that do not, or do not substantially self pair).
  • Rl and R3 each comprise an HLG-A molecule
  • R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.
  • Linker A and Linker B comprise Fc moieties (e.g., self pairing Fc moieties or Fc moieties that do not, or do not substantially self pair).
  • Rl and R3 each comprise an agonistic anti-LILRBl antibody molecule
  • R2 and R4 independentlycomprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.
  • Linker A and Linker B comprise Fc moieties (e.g., self pairing Fc moieties or Fc moieties that do not, or do not substantially self pair).
  • Rl and R3 each comprise an agonistic anti-KIR2DL4 antibody molecule
  • R2 and R4 independentlycomprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.
  • Linker A and Linker B comprise Fc moieties (e.g., self pairing Fc moieties or Fc moieties that do not, or do not substantially self pair).
  • Rl and R3 each comprise an agonistic anti-LILRB2 antibody molecule
  • R2 and R4 independentlycomprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.
  • Linker A and Linker B comprise Fc moieties (e.g., self pairing Fc moieties or Fc moieties that do not, or do not substantially self pair).
  • Rl and R3 each comprise an agonistic anti-NKG2A antibody molecule
  • R2 and R4 independentlycomprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.
  • Linker A and Linker B comprise Fc moieties (e.g., self pairing Fc moieties or Fc moieties that do not, or do not substantially self pair).
  • Rl and R3 comprises a first moiety chosen from, and the other comprises a different moiety chosen from: an antagonistic anti-LILRBl antibody molecule, an agonistic anti- KR2DL4 antibody molecule, and an agonistic anti-NKG2A antibody molecule; and
  • R2 and R4 independentlycomprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.
  • Linker A and Linker B comprise Fc moieties (e.g., self pairing Fc moieties or Fc moieties that do not, or do not substantially self pair).
  • Fc moieties e.g., self pairing Fc moieties or Fc moieties that do not, or do not substantially self pair.
  • Rl and R3 comprises an antagonistic anti-LILRBl antibody molecule and the other comprises an agonistic anti-KR2DL4 antibody molecule;
  • R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.
  • Linker A and Linker B comprise Fc moieties (e.g., self pairing Fc moieties or Fc moieties that do not, or do not substantially self pair).
  • Rl and R3 comprises an antagonistic anti-LILRBl antibody molecule and the other comprises an agonistic anti-NKG2A antibody molecule;
  • R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.
  • Linker A and Linker B comprise Fc moieties (e.g., self pairing Fc moieties or Fc moieties that do not, or do not substantially self pair).
  • Rl, R2, R3 and R4 each independently comprise: an IL-2 mutein molecule; a specific targeting moiety; or is absent;
  • an IL-2 mutein molecule and a specific targeting moiety are present.
  • Linker A and Linker B comprise Fc moieties (e.g., self pairing Fc moieties or Fc moieties that do not, or do not substantially self pair).
  • Rl, R2, R3 and R4 comprises an IL-2 mutein molecule, one comprises an anti- GITR antibody molecule, e.g., an anti-GITR antibody molecule that inhibits binding of GITRL to GITR, and one comprises a specific targeting moiety;
  • Linker A and Linker B comprise Fc moieties (e.g., self pairing Fc moieties or Fc moieties that do not, or do not substantially self pair).
  • Rl and R3 each comprise an IL-2 mutein molecule
  • R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.
  • Linker A and Linker B comprise Fc moieties (e.g., self pairing Fc moieties or Fc moieties that do not, or do not substantially self pair).
  • Fc moieties e.g., self pairing Fc moieties or Fc moieties that do not, or do not substantially self pair.
  • one of Rl and R3 comprises a GARP binding molecule, e.g., an anti-GARP antibody molecule or a GITR binding molecule, e.g., an anti-GITR antibody molecule and the other comprises an IL-2 mutein molecule; and
  • R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.
  • Linker A and Linker B comprise Fc moieties (e.g., self pairing Fc moieties or Fc moieties that do not, or do not substantially self pair).
  • one of Rl and R3 comprises a GARP binding molecule, e.g., an anti-GARP antibody molecule and the other comprises an IL-2 mutein molecule; and
  • R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.
  • Linker A and Linker B comprise Fc moieties (e.g., self pairing Fc moieties or Fc moieties that do not, or do not substantially self pair).
  • one of Rl and R3 comprises a GITR binding molecule, e.g., an anti-GITR antibody molecule, and the other comprises an IL-2 mutein molecule; and
  • R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.
  • Linker A and Linker B comprise Fc moieties (e.g., self pairing Fc moieties or Fc moieties that do not, or do not substantially self pair).
  • Rl, R2, R3 and R4 each independently comprise: an effector binding modulating moiety that activates an inhibitory receptor on a B cell, e.g., an anti-FCRL antibody molecule, e.g., an agonistic anti-FCRL antibody molecule; a specific targeting moiety; or is absent;
  • an effector binding moiety and a specific targeting moiety are present.
  • Linker A and Linker B comprise Fc moieties (e.g., self pairing Fc moieties or Fc moieties that do not, or do not substantially self pair).
  • the anti-FCRL molecule comprises: an anti-FCRL antibody molecule, e.g., an agonistic anti-FCRL antibody molecule, directed to FCRL1, FCRL2, FCRL3, FCRL4, FCRL5, or FCRL6.
  • Rl and R3 each comprises an agonistic anti-FCRL antibody molecule
  • R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.
  • Linker A and Linker B comprise Fc moieties (e.g., self pairing Fc moieties or Fc moieties that do not, or do not substantially self pair).
  • the anti-FCRL molecule comprises: an anti-FCRL antibody molecule, e.g., an agonistic anti-FCRL antibody molecule directed to FCRL1, FCRL2, FCRL3, FCRL4, FCRL5, or FCRL6.
  • an anti-FCRL antibody molecule e.g., an agonistic anti-FCRL antibody molecule directed to FCRL1, FCRL2, FCRL3, FCRL4, FCRL5, or FCRL6.
  • Rl and R3 independently comprise specific targeting moieties, e.g., antibody molecules against a tissue antigen
  • R2 and R4 each comprises an anti-FCRL antibody molecule, e.g., an agonistic anti-FCRL antibody molecule, e.g., an scFv molecule.
  • Linker A and Linker B comprise Fc moieties (e.g., self pairing Fc moieties or Fc moieties that do not, or do not substantially self pair).
  • the anti-FCRL molecule comprises: an anti-FCRL antibody molecule, e.g., an agonistic anti-FCRL antibody molecule directed to FCRL1, FCRL2, FCRL3, FCRL4, FCRL5, or FCRL6.
  • an anti-FCRL antibody molecule e.g., an agonistic anti-FCRL antibody molecule directed to FCRL1, FCRL2, FCRL3, FCRL4, FCRL5, or FCRL6.
  • Rl, R2, R3 and R4 comprises an anti-BCR antibody molecule, e.g., an antagonistic anti- BCR antibody molecule, one comprises an anti FCRL antibody molecule, and one comprises a specific targeting moiety.
  • an anti-BCR antibody molecule e.g., an antagonistic anti- BCR antibody molecule
  • one comprises an anti FCRL antibody molecule
  • one comprises a specific targeting moiety.
  • Linker A and Linker B comprise Fc moieties (e.g., self pairing Fc moieties or Fc moieties that do not, or do not substantially self pair).
  • the anti-FCRL molecule comprises: an anti-FCRL antibody molecule, e.g., an agonistic anti-FCRL antibody molecule directed to FCRL1, FCRL2, FCRL3, FCRL4, FCRL5, or FCRL6.
  • an anti-FCRL antibody molecule e.g., an agonistic anti-FCRL antibody molecule directed to FCRL1, FCRL2, FCRL3, FCRL4, FCRL5, or FCRL6.
  • Rl, R2, R3 and R4 comprises a bispecfic antibody molecule comprising an anti -BCR antibody molecule, e.g., an antagonistic anti- BCR antibody molecule, and an anti FCRL antibody molecule, and one comprises a specific targeting moiety;
  • Linker A and Linker B comprise Fc moieties (e.g., self pairing Fc moieties or Fc moieties that do not, or do not substantially self pair).
  • the anti-FCRL molecule comprises: an anti-FCRL antibody molecule, e.g., an agonistic anti-FCRL antibody molecule directed to FCRL1, FCRL2, FCRL3, FCRL4, FCRL5, or FCRL6.
  • an anti-FCRL antibody molecule e.g., an agonistic anti-FCRL antibody molecule directed to FCRL1, FCRL2, FCRL3, FCRL4, FCRL5, or FCRL6.
  • Rl, R2, R3 and R4 each independently comprise:
  • an effector binding/modulating moiety e.g., an ICIM binding/modulating moiety, an IIC binding/modulating moiety, ICSM binding/modulating moiety, or an SM binding/modulating moiety, that minimizes or inhibits T cell activity, expansion, or function (a T cell effector binding/modulating moiety);
  • an effector binding/modulating moiety e.g., an ICIM binding/modulating moiety, an IIC binding/modulating moiety, ICSM binding/modulating moiety, or an SM binding/modulating moiety, that minimizes or inhibits B cell activity, expansion, or function (a B cell effector binding/modulating moiety);
  • T cell effector binding/modulating moiety a T cell effector binding/modulating moiety, a B cell effector binding/modulating moiety, and a specific targeting moiety are present.
  • Linker A and Linker B comprise Fc moieties (e.g., self pairing Fc moieties).
  • one of Rl, R2, R3, and R4 comprises an agonistic anti-PD-l antibody and one comprises an HLA-G molecule.
  • one of Rl, R2, R3, and R4 comprises an SM binding/modulating moiety, e.g., a CD39 molecule or a CD73 molecule.
  • one of Rl, R2, R3, and R4 comprises an entity that binds, activates, or maintains, a regulatory immune cell, e.g., a Treg cell or a Breg cell, for example, an IL-2 mutein molecule.
  • one of Rl, R2, R3, and R4 comprises an agonistic anti-PD-l antibody, or one comprises an HLA-G molecule, and one comprises an IL-2 mutein molecule.
  • the PD-l antibody is replaced with a IL-2 mutein molecule.
  • one of Rl, R2, R3, and R4 comprises an agonistic anti-PD-l antibody, one comprises an HLA-G molecule, and one comprises CD39 molecule or a CD73 molecule.
  • the PD-l antibody is replaced with a IL-2 mutein molecule.
  • linker Regions A and B can comprise Fc regions.
  • a therapeutic compound comprises a Linker Region that can self-associate.
  • a therapeutic compound comprises a Linker Region that has a moiety that minimizes self association, and typically Linker Region A and Linker Region B are heterodimers.
  • Linkers also include gly cine/ serine linkers.
  • the linker can comprise one or more repeats of GGGGS (SEQ ID NO: 23).
  • the linker comprises 1, 2, 3, 4, or 5 repeats of SEQ ID NO: 23.
  • the linker comprises or
  • linkers can be used in any of the therapeutic compounds or compositions provided herein.
  • the linker region can comprise a Fc region that has been modified (e.g . mutated) to produce a heterodimer.
  • the CH3 domain of the Fc region can be mutated.
  • Fc regions can be found in, for example, U.S. Patent No. 9,574,010, which is hereby incorporated by reference in its entirety.
  • the Fc region as defined herein comprises a CH3 domain or fragment thereof, and may additionally comprise one or more addition constant region domains, or fragments thereof, including hinge, CH1, or CH2.
  • the numbering of the Fc amino acid residues is that of the EU index as in Rabat et al., 1991, NIH Publication 91-3242, National Technical Information Service, Springfield, Va.
  • the "EU index as set forth in Rabat” refers to the EU index numbering of the human IgGl Rabat antibody.
  • 9,574,010 provides the amino acids numbered according to the EU index as set forth in Rabat of the CH2 and CH3 domain from human IgGl, which is hereby incorporated by reference.
  • Table 1.1 of U.S. Patent No. 9,574,010 provides mutations of variant Fc heterodimers that can be used as linker regions.
  • Table 1.1 of U.S. Patent No. 9,574,010 is hereby incorporated by reference.
  • the Linker Region A comprises a first CH3 domain polypeptide and a the Linker Region B comprises a second CH3 domain polypeptide, the first and second CH3 domain polypeptides independently comprising amino acid modifications as compared to a wild-type CH3 domain polypeptide, wherein the first CH3 domain polypeptide comprises amino acid modifications at positions T350, L351, F405, and Y407, and the second CH3 domain polypeptide comprises amino acid modifications at positions T350, T366, K392 and T394, wherein the amino acid modification at position T350 is T350V, T3501, T350L or T350M; the amino acid modification at position L351 is L351Y; the amino acid modification at position F405 is F405A, F405V, F405T or F405S; the amino acid modification at position Y407 is Y407V, Y407A or Y407I; the amino acid modification at position T366 is T366L, T366I, T366V
  • the amino acid modification at position K392 is K392M or K392L. In some embodiments, the amino acid modification at position T350 is T350V. In some embodiments, the first CH3 domain polypeptide further comprises one or more amino acid modifications selected from Q347R and one of S400R or S400E. In some embodiments, the second CH3 domain polypeptide further comprises one or more amino acid modifications selected from L351Y, K360E, and one of N390R, N390D or N390E.
  • the first CH3 domain polypeptide further comprises one or more amino acid modifications selected from Q347R and one of S400R or S400E
  • the second CH3 domain polypeptide further comprises one or more amino acid modifications selected from L351Y, K360E, and one of N390R, N390D or N390E.
  • the amino acid modification at position T350 is T350V.
  • the amino acid modification at position F405 is F405A.
  • the amino acid modification at position Y407 is Y407V.
  • the amino acid modification at position T366 is T366L or T366I.
  • the amino acid modification at position F405 is F405A
  • the amino acid modification at position Y407 is and Y407V
  • the amino acid modification at position T366 is T366L or T366I
  • the amino acid modification at position K392 is K392M or K392L.
  • the first CH3 domain polypeptide comprises the amino acid modifications T350V, L351Y, S400E, F405V and Y407V
  • the second CH3 domain polypeptide comprises the amino acid modifications T350V, T366L, N390R, K392M and T394W.
  • the first CH3 domain polypeptide comprises the amino acid modifications T350V, L351Y, S400E, F405T and Y407V
  • the second CH3 domain polypeptide comprises the amino acid modifications T350V, T366L, N390R, K392M and T394W
  • the first CH3 domain polypeptide comprises the amino acid modifications T350V, L351Y, S400E, F405S and Y407V
  • the second CH3 domain polypeptide comprises the amino acid modifications T350V, T366L, N390R, K392M and T394W.
  • the first CH3 domain polypeptide comprises the amino acid modifications T350V, L351Y, S400E, F405A and Y407V
  • the second CH3 domain polypeptide comprises the amino acid modifications T350V, L351Y, T366L, N390R, K392M and T394W
  • the first CH3 domain polypeptide comprises the amino acid modifications Q347R, T350V, L351Y, S400E, F405A and Y407V
  • the second CH3 domain polypeptide comprises the amino acid modifications T350V, K360E, T366L, N390R, K392M and T394W.
  • the first CH3 domain polypeptide comprises the amino acid modifications T350V, L351Y, S400R, F405A and Y407V
  • the second CH3 domain polypeptide comprises the amino acid modifications T350V, T366L, N390D, K392M and T394W
  • the first CH3 domain polypeptide comprises the amino acid modifications T350V, L351Y, S400R, F405A and Y407V
  • the second CH3 domain polypeptide comprises the amino acid modifications T350V, T366L, N390E, K392M and T394W.
  • the first CH3 domain polypeptide comprises the amino acid modifications T350V, L351Y, S400E, F405A and Y407V
  • the second CH3 domain polypeptide comprises the amino acid modifications T350V, T366L, N390R, K392L and T394W
  • the first CH3 domain polypeptide comprises the amino acid modifications T350V, L351Y, S400E, F405A and Y407V
  • the second CH3 domain polypeptide comprises the amino acid modifications T350V, T366L, N390R, K392F and T394W.
  • an isolated heteromultimer comprising a heterodimeric CH3 domain comprising a first CH3 domain polypeptide and a second CH3 domain polypeptide, the first CH3 domain polypeptide comprising amino acid modifications at positions F405 and Y407, and the second CH3 domain polypeptide comprising amino acid modifications at positions T366 and T394, wherein: (i) the first CH3 domain polypeptide further comprises an amino acid modification at position L351, and (ii) the second CH3 domain polypeptide further comprises an amino acid modification at position K392, wherein the amino acid modification at position F405 is F405A, F405T, F405S or F405V; and the amino acid modification at position Y407 is Y407V, Y407A, Y407L or Y407I; the amino acid modification at position T394 is T394W; the amino acid modification at position L351 is L351Y; the amino acid modification at position K392 is K392L, K392L, K39
  • the Linker Region A comprises a first CH3 domain polypeptide and a t Linker Region B comprises a second CH3 domain polypeptide, wherein the first CH3 domain polypeptide comprising amino acid modifications at positions F405 and Y407, and the second CH3 domain polypeptide comprising amino acid modifications at positions T366 and T394, wherein: (i) the first CH3 domain polypeptide further comprises an amino acid
  • the second CH3 domain polypeptide further comprises an amino acid modification at position K392, wherein the amino acid modification at position F405 is F405A, F405T, F405S or F405V; and the amino acid modification at position Y407 is Y407V, Y407A, Y407L or Y407I; the amino acid modification at position T394 is T394W; the amino acid modification at position L351 is L351Y; the amino acid modification at position K392 is K392L, K392M, K392V or K392F, and the amino acid modification at position T366 is T366I, T366L, T366M or T366V, wherein the heterodimeric CH3 domain has a melting temperature (Tm) of about 70 C.
  • Tm melting temperature
  • the amino acid modification at position F405 is F405A.
  • the amino acid modification at position T366 is T366I or T366L.
  • the amino acid modification at position Y407 is Y407V.
  • the amino acid modification at position F405 is F405A, the amino acid modification at position Y407 is Y407V, the amino acid modification at position T366 is T366I or T366L, and the amino acid modification at position K392 is K392L or K392M.
  • the amino acid modification at position F405 is F405A
  • the amino acid modification at position Y407 is Y407V
  • the amino acid modification at position T366 is T366L
  • the amino acid modification at position K392 is K392M.
  • the amino acid modification at position F405 is F405A
  • the amino acid modification at position Y407 is Y407V
  • the amino acid modification at position T366 is T366L
  • the amino acid modification at position K392 is K392L.
  • the amino acid modification at position F405 is F405A
  • the amino acid modification at position Y407 is Y407V
  • the amino acid modification at position T366 is T366I
  • the amino acid modification at position K392 is K392M.
  • the amino acid modification at position F405 is F405A
  • the amino acid modification at position Y407 is Y407V
  • the amino acid modification at position T366 is T366I
  • the amino acid modification at position K392 is K392L
  • the first CH3 domain polypeptide further comprises an amino acid modification at position S400 selected from S400D and S400E
  • the second CH3 domain polypeptide further comprises the amino acid modification N390R.
  • the amino acid modification at position F405 is F405A
  • the amino acid modification at position Y407 is Y405V
  • the amino acid modification at position S400 is S400E
  • the amino acid modification at position T366 is T366L
  • the amino acid modification at position K392 is K392M.
  • the modified first and second CH3 domains are comprised by an Fc construct based on a type G immunoglobulin (IgG).
  • IgG immunoglobulin
  • the IgG can be an IgGl, IgG2, IgG3 or IgG4.
  • Linker Region A and Linger Region B comprising variant CH3 domains are described in U.S. Patent Nos. 9,499,634 and 9,562,109, each of which is incorporated by reference in its entirety.
  • a Linker Region A and Linker Region B can be complementary fragments of a protein, e.g., a naturally occurring protein such as human serum albumin.
  • one of Linker Region A and Linker Region B comprises a first, e.g., an N terminal fragment of the protein, e.g., hSA, and the other comprises a second, e.g., a C terminal fragment of the protein, e.g., has.
  • the fragments comprise an N terminal and a C terminal fragment.
  • the fragments comprise two internal fragments. Typically the fragments do not overlap.
  • the first fragment provides a N terminus and a C terminus for linking, e.g., fusing, to other sequences, e.g., sequences of Rl, R2, R3, or R4 (as defined herein).
  • the Linker Region A and the Linker Region B can be derived from albumin polypeptide.
  • the albumin polypeptide is selected from native human serum albumin polypeptide and human alloalbumin polypeptide.
  • the albumin polypeptide can be modified such that the Linker Region A and Linker Region B interact with one another to form heterodimers. Examples of modified albumin polypeptides are described in U.S. Patent Nos. 9,388,231 and 9,499,605, each of which is hereby incorporated by reference in its entirety.
  • the Linker Region A comprises a first polypeptide and the Linker Region B comprises a second polypeptide; wherein each of said first and second polypeptides comprises an amino acid sequence comprising a segment of an albumin polypeptide selected from native human serum albumin polypeptide and human alloalbumin polypeptide; wherein said first and second polypeptides are obtained by
  • said first polypeptide comprises at least one mutation selected from A194C, L198C, W214C, A217C, L331C and A335C
  • said second polypeptide comprises at least one mutation selected from L331C, A335C, V343C, L346C, A350C, V455C, and N458C; and wherein said first and second polypeptides self-assemble to form a quasi-native structure of the monomeric form of the albumin polypeptide.
  • the segmentation site resides on a loop of the albumin polypeptide that has a high solvent accessible surface area (SASA) and limited contact with the rest of the albumin structure, b) the segmentation results in a complementary interface between the transporter polypeptides.
  • SASA solvent accessible surface area
  • the first polypeptide comprises residues 1-337 or residues 1-293 of the albumin polypeptide with one or more of the mutations described herein.
  • the second polypeptide comprises residues of 342-585 or 304-585 of the albumin polypeptide with one or more of the mutations described herein.
  • the first polypeptide comprises residues 1-339, 1-300, 1-364, 1-441, 1-83, 1-171, 1-281, 1-293, 1-114, 1- 337, or 1-336 of the albumin protein.
  • the second polypeptide comprises residues 301-585, 365-585, 442-585, 85-585, 172-585, 282-585, or 115-585, 304-585, 340-585, or 342-585 of the albumin protein.
  • the first and second polypeptide comprise the residues of the albumin protein as shown in the table below.
  • the sequence of the albumin protein is described below.
  • the first and second polypeptides comprise a linker that can form a covalent bond with one another, such as a disulfide bond.
  • a non-limiting example of the linker is a peptide linker.
  • the peptide linker comprises GGGGS.
  • the linker can be fused to the C-terminus of the first polypeptide and the N-terminus of the second polypeptide.
  • the linker can also be used to attach the moieties described herein without abrogating the ability of the linkers to form a disulfide bond.
  • the first and second polypeptides do not comprise a linker that can form a covalent bond.
  • the first and second polypeptides have the following substitutions.
  • the sequence of the albumin polypeptide can be The sequence of human albumin is as shown, in the post-protein form with the N-terminal signaling residues removed
  • the Linker Region A and the Linker Region B form a heterodimer as described herein.
  • the polypetide comprises at the N-terminus an antibody comprised of F(ab’)2 on an IgGl Fc backbone fused with scFvs on the C-terminus of the IgG Fc backbone.
  • the IgG Fc backbone is a IgGl Fc backbone.
  • the IgGl backbone is replaced with a IgG4 backbone, IgG2 backbone, or other similar IgG backbone.
  • the IgG backbones described in this paragraph can be used throughout this application where a Fc region is referred to as part of the therapeutic compound.
  • the antibody comprised of F(ab’)2 on an IgGl Fc backbone can be an anti- MAdCAM antibody or an anti -PD- 1 antibody on an IgGl Fc or any other targeting moiety or effector binding/modulating moiety provided herein.
  • the The scFV segments fused to the C-terminus could be an anti-PD-l antibody, if the N-terminus region is an anti-MAdCAM antibody, or anti-MAdCAM antibody, if the N-terminus region is an anti-PD-l antibody.
  • the N-terminus can be the targeting moiety, such as any one of the ones provided for herein, and the C-terminus can be the effector binding/modulating moiety, such as any of the ones provided for herein.
  • the N- terminus can be the effector binding/modulating moiety, such as any one of the ones provided for herein
  • the C-terminus can be the targeting moiety, such as any of the ones provided for herein.
  • the N-terminus can be the targeting moiety, such as any one of the ones provided for herein, and the C-terminus can be the effector binding/modulating moiety, such as any of the ones provided for herein.
  • the therapeutic compound comprises two polypeptides that homodimerize.
  • the N-terminus of the polypeptide comprises an effector binding/modulating moiety that is fused to a human IgGl Fc domain (e.g. CH2 and/or CH3 domains).
  • the C-terminus of the Fc domain is another linker that is fused to the targeting moiety.
  • the molecule could be represented using the formula of Rl-Linker A-Fc Region-Linker B-R2, wherein Rl can be an effector
  • R2 is a targeting moiety
  • Linker A and Linker B are independently linkers as provided for herein.
  • Linker 1 and Linker 2 are different.
  • the molecule could be represented using the formula of Rl-Linker A-Fc Region-Linker B-R2, wherein Rl can be a targeting moiety, R2 is an effector
  • Linker A and Linker B are independently linkers as provided for herein. In some embodiments, Linker A and Linker B are different. The linkers can be chosen from the non-limiting exmaples provided for herein.
  • Rl and R2 are independently selected from F(ab’)2 and scFV antibody domains. In some embodiments, Rl and R2 are different antibody domains. In some embodiments, the scFV is in the VL-VH domain orientation.
  • the therapeutic compound is a bispecific antibody.
  • the bispecific antibodies are comprised of four polypeptide chains comprising the following: Chain 1 : nt-VHl-CHl-CH2-CH3 -Linker A-scFv[VL2-Linker B-VH2]-ct
  • Chain 2 nt-VHl-CHl-CH2-CH3 -Linker A-scFv[VL2-Linker B-VH2]-ct
  • chains 1 and 2 are identical to eachother, and chains 3 and 4 are identical to each other,
  • Linker A comprises GGGGS (SEQ ID NO: 23), or two repeats thereof,
  • Linker B comprises GGGGS (SEQ ID NO: 23), or two repets thereof, GGGGS GGGGS GGGGS (SEQ ID NO: 30), or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 22).
  • the scFv may be arranged in the NT-VH2-VL2-CT or NT-VL2-VH2-CT orientation.
  • NT or nt stands for N-terminus
  • CT or ct stands for C-terminus of the protein.
  • CH1, CH2, and CH3 are the domains from the IgG Fc region, and CL stands for Constant Light chain, which can be either kappa or lambda family light chains. The other definitions stand for the way they are normally used in the art.
  • the VH1 and VL1 domains are derived from the effector molecule and the VH2 and VL2 domains are derived from the targeting moiety. In some embodiments the VH1 and VL1 domains are derived from a targeting moiety and the VH2 and VL2 domains are derived from an effector binding/modulating moiety.
  • the VH1 and VL1 domains are derived from an anti-PD-l antibody, and the VH2 and VL2 domains are derived from an anti-MAdCAM antibody. In some embodiments the VH1 and VL1 domains are derived from an anti-MAdCAM antibody and the VH2 and VL2 domains are derived from an anti-PD-l antibody.
  • Linker A comprises 1, 2, 3, 4, or 5 GGGGS (SEQ ID NO: 23) repeats.
  • Linker B comprises 1, 2, 3, 4, or 5 GGGGS (SEQ ID NO: 23) repeats.
  • Linker A and Linker B can be the same or different.
  • Linker A comprises GGGGS (SEQ ID NO: 23), or two repeats thereof, GGGGS GGGGS GGGGS (SEQ ID NO: 30), or GGGGS GGGGS GGGGS (SEQ ID NO: 22).
  • Linker B comprises GGGGS (SEQ ID NO: 23), or two repeats thereof, GGGGSGGGGSGGGGS (SEQ ID NO: 30), or GGGGS GGGGS GGGGS GGGGS (SEQ ID NO: 22).
  • the therapeutic compound comprises a light chain and a heavy chain.
  • the light and heavy chain begin at the N-terminus with the VH domain of a targeting moiety followed by the CH1 domain of a human IgGl, which is fused to a Fc region ( e.g . CH2-CH3) of human IgGl.
  • a linker as provided herein, such as but not limited to, GGGGS (SEQ ID NO: 23), or two or three repeats thereof, or GGGGSGGGGSGGGGS (SEQ ID NO: 22).
  • the linekr can then be fused to an effector binding/modulating moiety, such as any one of the effector moieties provided for herein.
  • the polypeptides can homodimerize because through the heavy chain homodimerization, which results in a therapeutic compound having two effector moieties, such as two anti-PD-l antibodies.
  • the targeting moiety is an IgG format, there are two Fab arms that each recognize binding partner of the targeting moiety, for example, MAdCAM being bound by the anti-MAdCAM targeting moiety.
  • the Fc domain bears mutations to render the Fc region“effectorless,” that is unable to bind FcRs.
  • the mutations that render Fc regions effectorless are known.
  • the mutations in the Fc region which is according to the known numbering system, are selected from the group consisting of: K322A, L234A, L235A, G237A, L234F, L235E, N297, P331S, or any combination thereof.
  • the Fc mutations comprises a mutation at L234 and/or L235 and/or G237.
  • the Fc mutations comprise L234A and/or L235A mutations, which can be referred to as LALA mutations.
  • the Fc mutations comprise L234A, L235A, and G237A mutations.
  • Therapeutic compounds can comprise a plurality of specific targeting moieties.
  • the therapeutic compound comprises a plurality one specific targeting moiety, a plurality of copies of a donor specific targeting moiety or a plurality of tissue specific targeting moieties.
  • a therapeutic compound comprises a first and a second donor specific targeting moiety, e.g., a first donor specific targeting moiety specific for a first donor target and a second donor specific targeting moiety specific for a second donor target, e.g., wherein the first and second target are found on the same donor tissue.
  • the therapeutic compound comprises e.g., a first specific targeting moiety for a tissue specific target and a second specific targeting moiety for a second target, e.g., wherein the first and second target are found on the same or different target tissue,
  • a therapeutic compound comprises a plurality of effector binding/modulating moieties each comprising an ICIM binding/modulating moiety, the number of ICIM binding/modulating moieties is sufficiently low that clustering of the ICIM
  • binding/modulating moiety s ligand on immune cells (in the absence of target binding) is minimized, e.g., to avoid systemic agonizing of immune cells in the absence of binding of the therapeutic compound to target.
  • a component of a therapeutic molecule is derived from or based on a reference molecule, e.g., in the case of a therapeutic molecule for use in humans, from a naturally occurring human polypeptide.
  • a reference molecule e.g., in the case of a therapeutic molecule for use in humans, from a naturally occurring human polypeptide.
  • binding/modulating moiety an inhibitory immune checkpoint molecule ligand molecule, an inhibitory molecule counter ligand molecule, a SM binding/modulating moiety, a specific targeting moiety, a target ligand binding molecule, or a tissue specific targeting moiety, can be based on or derived from a naturally occurring human polypeptide.
  • a PD-L1 molecule can be based on or derived from a human PD-L1 sequence.
  • a therapeutic compound component e.g., a PD-Ll molecule: a) comprises all or a portion of, e.g., an active portion of, a naturally occurring form of the human polypeptide;
  • b) comprises all or a portion of, e.g., an active portion of, a human polypeptide having a sequence appearing in a database, e.g., GenBank database, on January 11, 2017, a naturally occurring form of the human polypeptide that is not associated with a disease state ;
  • c) comprises a human polypeptide having a sequence that differs by no more than 1, 2, 3, 4, 5, 10, 20, or 30 amino acid residues from a sequence of a) or b);
  • d) comprises a human polypeptide having a sequence that differs at no more than by 1, 2, 3, 4, 5 10, 20, or 30 % its amino acids residues from a sequence of a) or b);
  • e) comprises a human polypeptide having a sequence that does not differ substantially from a sequence of a) or b); or
  • f) comprises a human polypeptide having a sequence of c), d),or e) that does not differ substantially in a biological activity, e.g., ability to enhance or inhibit an immune response, from a human polypeptide having the sequence of a) or b).
  • therapeutic compounds can comprise a plurality of effector binding/modulating moieties.
  • a therapeutic compound can comprise two or more of the following selected from:
  • a therapeutic compound can comprise a plurality, e.g., two, ICIM binding/modulating moieties (wherein they are the same or different); by way of example, two that activate or agonize PD-l; a plurality, e.g., two, IIC binding/modulating moieties; (wherein they are the same or different); a plurality, e.g., two, SM binding/modulating moieties (wherein they are the same or different), or a plurality, e.g., tow, ICSM binding/modulating moieties (wherein they are the same or different).
  • the therapeutic compound can comprise an ICIM binding/modulating moiety and an IIC binding/modulating moiety; an ICIM binding/modulating moiety and an SM binding/modulating moiety; an IIC binding/modulating moiety and an SM binding/modulating moiety, an ICIM binding/modulating moiety and an ICSM binding/modulating moiety; an IIC binding/modulating moiety and an ICSM
  • the therapeutic compound comprises a plurality of targeting moieties.
  • the targeting moieties can be the same or different.
  • compositions e.g., pharmaceutically acceptable compositions, which include a therapeutic compound described herein, formulated together with a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • the carrier can be suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, local, ophthalmic, topical, spinal or epidermal administration (e.g. by injection or infusion).
  • the term“carrier” means a diluent, adjuvant, or excipient with which a compound is administered.
  • pharmaceutical carriers can also be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
  • the pharmaceutical carriers can also be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like.
  • auxiliary, stabilizing, thickening, lubricating and coloring agents can be used.
  • the carriers can be used in pharmaceutical compositions comprising the therapeutic compounds provided for herein.
  • compositions and compounds of the embodiments provided for herein may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, liposomes and suppositories.
  • liquid solutions e.g., injectable and infusible solutions
  • dispersions or suspensions e.g., dispersions or suspensions
  • liposomes and suppositories e.g., liposomes and suppositories.
  • Typical compositions are in the form of injectable or infusible solutions.
  • the mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular).
  • the therapeutic molecule is administered by intravenous infusion or injection.
  • the therapeutic molecule is administered by intramuscular or subcutaneous injection.
  • the therapeutic molecule is administered locally, e.g., by injection, or topical application,
  • parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.
  • compositions typically should be sterile and stable under the conditions of manufacture and storage.
  • the composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable to high therapeutic molecule
  • Sterile injectable solutions can be prepared by incorporating the active compound (i.e., therapeutic molecule) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those
  • sterile powders for the preparation of sterile injectable solutions the preferred methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.
  • the proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.
  • the active compound may be prepared with a carrier that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems.
  • a controlled release formulation including implants, transdermal patches, and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
  • a therapeutic compound can be orally administered, for example, with an inert diluent or an assimilable edible carrier.
  • the compound (and other ingredients, if desired) may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the subject's diet.
  • the compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • To administer a compound by other than parenteral administration it may be necessary to coat the compound with, or co- administer the compound with, a material to prevent its inactivation.
  • Therapeutic compositions can also be administered with medical devices known in the art.
  • Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.
  • an exemplary, non-limiting range for a therapeutically or prophylactically effective amount of a therapeutic compound is 0.1-30 mg/kg, more preferably 1-25 mg/kg. Dosages and therapeutic regimens of the therapeutic compound can be determined by a skilled artisan.
  • the therapeutic compound is administered by injection (e.g., subcutaneously or intravenously) at a dose of about 1 to 40 mg/kg, e.g., 1 to 30 mg/kg, e.g., about 5 to 25 mg/kg, about 10 to 20 mg/kg, about 1 to 5 mg/kg, 1 to 10 mg/kg, 5 to 15 mg/kg, 10 to 20 mg/kg, 15 to 25 mg/kg, or about 3 mg/kg.
  • the dosing schedule can vary from e.g., once a week to once every 2, 3, or 4 weeks.
  • the therapeutic compound is administered at a dose from about 10 to 20 mg/kg every other week.
  • the therapeutic compound can be administered by intravenous infusion at a rate of more than 20 mg/min, e.g., 20-40 mg/min, and typically greater than or equal to 40 mg/min to reach a dose of about 35 to 440 mg/m2, typically about 70 to 310 mg/m2, and more typically, about 110 to 130 mg/m2.
  • the infusion rate of about 110 to 130 mg/m2 achieves a level of about 3 mg/kg.
  • the therapeutic compound can be administered by intravenous infusion at a rate of less than 10 mg/min, e.g., less than or equal to 5 mg/min to reach a dose of about 1 to 100 mg/m2, e.g., about 5 to 50 mg/m2, about 7 to 25 mg/m2, or, about 10 mg/m2.
  • the therapeutic compound is infused over a period of about 30 min. It is to be noted that dosage values may vary with the type and severity of the condition to be alleviated.
  • compositions may include a "therapeutically effective amount" or a
  • prophylactically effective amount of a therapeutic molecule.
  • a “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result.
  • a therapeutically effective amount of a therapeutic molecule may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the therapeutic compound to elicit a desired response in the individual.
  • therapeutically effective amount is also one in which any toxic or detrimental effects of a therapeutic molecule t is outweighed by the therapeutically beneficial effects.
  • a "therapeutically effective dosage" preferably inhibits a measurable parameter, e.g., immune attack at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and still more preferably by at least about 80% relative to untreated subjects.
  • the ability of a compound to inhibit a measurable parameter, e.g., immune attack can be evaluated in an animal model system predictive of efficacy in transplant rejection or autoimmune disorders.
  • composition can be evaluated by examining the ability of the compound to inhibit, such inhibition in vitro by assays known to the skilled practitioner.
  • prophylactically effective amount refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.
  • kits comprising a therapeutic compound described herein.
  • the kit can include one or more other elements including: instructions for use; other reagents, e.g., a label, a therapeutic agent, or an agent useful for chelating, or otherwise coupling, a therapeutic molecule to a label or other therapeutic agent, or a radioprotective composition; devices or other materials for preparing the a therapeutic molecule for
  • embodiments provided herein also include, but are not limited to:
  • a therapeutic compound comprising:
  • a specific targeting moiety selected from:
  • a donor specific targeting moiety which, e.g., preferentially binds a donor target
  • tissue specific targeting moiety which, e.g., preferentially binds target tissue of a subject
  • an effector binding/modulating moiety selected from:
  • an immune cell inhibitory molecule binding/modulating moiety (a) an immune cell inhibitory molecule binding/modulating moiety (ICIM binding/modulating moiety);
  • an immunosuppressive immune cell binding/modulating moiety (b) an immunosuppressive immune cell binding/modulating moiety (IIC binding/modulating moiety); or
  • an effector binding/modulating moiety that, as part of a therapeutic compound, promotes an immuno-suppressive local microenvironment, e.g., by providing in the proximity of the target, a substance that inhibits or minimizes attack by the immune system of the target (SM binding/modulating moiety).
  • binding/modulating moiety is expressed by an immune cell.
  • the therapeutic compound of embodiment 1, wherein the effector binding/modulating moiety comprises an ICIM binding/modulating moiety. 16. The therapeutic compound of embodiment 15, wherein the effector binding/modulating moiety comprises an ICIM binding/modulating moiety comprising an inhibitory immune checkpoint molecule ligand molecule.
  • inhibitory immune molecule counter-ligand molecule comprises a PD-L1 molecule.
  • ICIM is wherein the inhibitory immune molecule counter ligand molecule engages a cognate inhibitory immune checkpoint molecule selected from PD-l, KIR2DL4, LILRB1, LILRB, or CTLA-4.
  • the therapeutic compound of embodiment 18, wherein the ICIM comprises an antibody that binds to PD-l, KIR2DL4, LILRB 1, LILRB, or CTLA-4.
  • ICIM is wherein the inhibitory immune molecule counter ligand molecule engages a cognate inhibitory immune checkpoint molecule selected from PD-l, KIR2DL4, LILRB 1, LILRB, or CTLA-4.
  • the effector binding/modulating moiety comprises a ICIM binding/modulating moiety which comprises a functional antibody molecule to a cell surface inhibitory molecule.
  • binding/modulating moiety binds a different target than the effector binding/modulating moiety.
  • the effector binding/modulating moiety comprises an IIC binding/modulating moiety, which, increases, recruits or accumulates an immunosuppressive immune cell at the target site.
  • the effector binding/modulating moiety comprises a cell surface molecule binder which binds or specifically binds, a cell surface molecule on an immunosuppressive immune cell.
  • the therapeutic compound of embodiment 1, wherein the effector binding/modulating moiety comprises a cell surface molecule ligand molecule that binds or specifically binds, a cell surface molecule on an immunosuppressive immune cell.
  • immunosuppressive immune cell comprises a T regulatory cell, such as a a Foxp3+CD25+ T regulatory cell.
  • the therapeutic compound of any of embodiments 44 and 45, wherein the effector molecule binding moiety increases the availability, e.g., by increasing the local concentration or amount, of a substance which inhibits immune cell function, e.g., a substance that inhibits the activation of an immune cell or the function of an activated immune cell.
  • the therapeutic compound of any of embodiments 44-47, wherein the effector molecule binding moiety decreases the availability, e.g., by decreasing the local concentration or amount, or sequestering, of a substance which promotes immune cell function, e.g., a substance that promotes the activation of an immune cell or the function of an activated immune cell.
  • a substance which promotes immune cell function e.g., a substance that promotes the activation of an immune cell or the function of an activated immune cell.
  • binding/modulating moiety promotes an immuno-suppressive local microenvironment, e.g., by providing in the proximity of the target, a substance that inhibits or minimizes attack by the immune system of the target.
  • binding/modulating moiety comprises a molecule that inhibits or minimizes attack by the immune system of the target.
  • binding/modulating moiety binds and/or accumulate a soluble substance, e.g., an endogenous or exogenous substance having immunosuppressive function.
  • binding/modulating moiety binds and/or inhibits, sequesters, degrades or otherwise neutralizes a substance, e.g., a soluble substance, typically and endogenous soluble substance, that promotes immune attack.
  • the therapeutic compound any one of embodiments 44-52, wherein the effector molecule binding moiety decreases the availability of ATP or AMP.
  • binding/modulating moiety binds, or comprises, a substance, e.g., CD39 or CD73, that depletes a component that promotes immune effector cell function, e.g., ATP or AMP.
  • binding/modulating moiety comprises a CD39 molecule.
  • binding/modulating moiety comprises a CD73 molecule.
  • binding/modulating moiety comprises an anti-CD39 molecule.
  • binding/modulating moiety comprises an anti-CD73 antibody molecule.
  • the therapeutic compound any one of embodiments 44-54, wherein the effector molecule binding moiety comprises an immune-suprressive substance, e.g. a fragment an
  • binding/modulating moiety comprises alkaline phosphatase molecule.
  • Rl, R2, R3, and R4, each independently comprises an effector
  • binding/modulating moiety e.g., an ICIM binding/modulating moiety, an IIC
  • binding/modulating moiety or an SM binding/modulating moiety; a specific targeting moiety; or is absent; provided that an effector binding/modulating moiety and a specific targeting moiety are present.
  • each of Linker Region A and Linker Region B comprises an Fc region.
  • the therapeutic compound of any of embodiments 61-68, wherein the linker is a glycine/serine linker, such as 1, 2, 3, 4, or 5 repeats of GGGGS (SEQ ID NO: 23).
  • the linker comprises a Fc region and a glycine/serine linker, such as 1, 2, 3, 4, or 5 repeats of GGGGS (SEQ ID NO: 23).
  • Rl and R3 independently comprise a functional anti-PD-l antibody molecule (an agonist of PD-l); and R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.
  • Rl and R3 independently comprise specific targeting moieties, e.g., an anti-tissue antigen antibody; and R2 and R4 independently comprise a functional anti-PD-l antibody molecule (an agonist of PD-l).
  • Rl, R2, R3 and R4 each independently comprise: an SM binding/modulating moiety which modulates, e.g., binds and inhibits, sequesters, degrades or otherwise neutralizes a substance, e.g., a soluble molecule that modulates an immune response, e.g., ATP or AMP, e.g., a CD39 molecule or a CD73 molecule; a specific targeting moiety; or is absent;
  • an SM binding/modulating moiety which modulates, e.g., binds and inhibits, sequesters, degrades or otherwise neutralizes a substance, e.g., a soluble molecule that modulates an immune response, e.g., ATP or AMP, e.g., a CD39 molecule or a CD73 molecule; a specific targeting moiety; or is absent;
  • an SM binding/modulating moiety and a specific targeting moiety are present.
  • Rl and R3 independently comprise a CD39 molecule or a CD73 molecule
  • R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.
  • Rl and R3 each comprises a CD39 molecule
  • R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.
  • Rl and R3 each comprises a CD73 molecule
  • R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen. 80.
  • Rl and R3 comprises a CD39 molecule and the other comprises a CD73 molecule; and R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.
  • Rl, R2, R3 and R4 each independently comprise: an HLA-G molecule; a specific targeting moiety; or is absent;
  • HLA-G molecule and a specific targeting moiety are present.
  • Rl and R3 each comprise an HLG-A molecule
  • R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.
  • Linker A and Linker B comprise Fc moieties (e.g., self pairing Fc moieties or Fc moieties that do not, or do not substantially self pair).
  • Rl and R3 each comprise an agonistic anti-LILRBl antibody molecule
  • R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.
  • Linker A and Linker B comprise Fc moieties (e.g., self pairing Fc moieties or Fc moieties that do not, or do not substantially self pair).
  • Rl and R3 each comprise an agonistic anti-KIR2DL4 antibody molecule
  • R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.
  • Linker A and Linker B comprise Fc moieties (e.g., self pairing Fc moieties or Fc moieties that do not, or do not substantially self pair).
  • Rl and R3 each comprise an agonistic anti-LILRB2 antibody molecule
  • R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.
  • Rl and R3 each comprise an agonistic anti-NKG2A antibody molecule
  • R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.
  • Rl and R3 comprises a first moiety chosen from, and the other comprises a different moiety chosen from: an antagonistic anti-LILRBl antibody molecule, an agonistic anti- KR2DL4 antibody molecule, and an agonistic anti-NKG2A antibody molecule; and
  • R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.
  • Rl and R3 comprises an antagonistic anti-LILRBl antibody molecule and the other comprises an agonistic anti-KR2DL4 antibody molecule;
  • R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.
  • Rl and R3 comprises an antagonistic anti-LILRBl antibody molecule and the other comprises an agonistic anti-NKG2A antibody molecule;
  • R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.
  • Rl, R2, R3 and R4 each independently comprise: an IL-2 mutein molecule; a specific targeting moiety; or is absent; and
  • Rl and R3 each comprise an IL-2 mutein molecule
  • R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.
  • Rl and R3 comprises a MAdCAM binding molecule, e.g., an anti- MAdCAM antibody molecule or a GITR binding molecule, e.g., an anti-GITR antibody molecule and the other comprises an IL-2 mutein molecule; and R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.
  • a MAdCAM binding molecule e.g., an anti- MAdCAM antibody molecule or a GITR binding molecule, e.g., an anti-GITR antibody molecule and the other comprises an IL-2 mutein molecule
  • R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.
  • one of Rl and R3 comprises a GARP binding molecule, e.g., an anti-GARP antibody molecule and the other comprises an IL-2 mutein molecule; and
  • R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.
  • one of Rl and R3 comprises a GARP binding molecule, e.g., an anti-GARP antibody molecule or a GITR binding molecule, e.g., an anti-GITR antibody molecule and the other comprises an IL-2 mutein molecule; and
  • R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.
  • one of Rl and R3 comprises a GARP binding molecule, e.g., an anti-GARP antibody molecule and the other comprises an IL-2 mutein molecule; and
  • R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.
  • one of Rl and R3 comprises a GITR binding molecule, e.g., an anti-GITR antibody molecule, and the other comprises an IL-2 mutein molecule; and
  • R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.
  • IL-2 mutein IL-2 mutant polypeptide
  • the therapeutic compound of embodiment 89H, wherein the targeting moiety comprises an antibody that binds to a target protein on the surface of a target cell.
  • the antibody is an antibody that binds to MAdCAM, OAT1 (SLC22A6), OCT2 (SLC22A2), FXYD2, TSPAN7, DPP6,
  • HEPACAM2 HEPACAM2, TMEM27, or GPR119.
  • 89M The therapeutic compound of any of embodiments 89H-89L, wherein the immune cell causes a disease pathology.
  • 89N The therapeutic compound of any of embodiments 89H-89M, wherein the targeting moiety comprises an anti-MAdCAM antibody.
  • Rl, R2, R3, and R4, each independently comprises the effector
  • binding/modulating moiety the targeting moiety, or is absent.
  • 89R The therapeutic compound of embodiments 890, 89P, or 89Q, wherein Rl is the IL- mutein and R2 is an anti-MAdCAM antibody.
  • 89T The therapeutic compound of embodiments 890, 89P, or 89Q, wherein one of R3 and R4 is the IL-2 mutein and one of R3 and R4 is an anti-MAdCAM antibody.
  • 89V The therapeutic compound of embodiments 890, 89P, or 89Q, wherein R3 is an anti- MAdCAM antibody and one R4 is the IL-2 mutein. 89W.
  • 89X The therapeutic compound of any of embodiments 890-89W, wherein the linker comprises a sequence of GGGGSGGGGSGGGGSGGGGS, GGGGS GGGGS GGGGS ,
  • GGGGSGGGGS GGGGSGGGGS, or GGGGS.
  • IL-2 mutein comprises a IL- 2 sequence of SEQ ID NO: 6, wherein peptide comprises a mutation at a position that corresponds to position 53, 56, 80, or 118 of SEQ ID NO: 6.
  • IL-2 mutein comprises a IL-2 sequence of SEQ ID NO: 6, wherein peptide comprises a mutation at a position that corresponds to position 53, 56, 80, or 118 of SEQ ID NO: 6.
  • 89BB The therapeutic compound of embodiment 89Z, wherein the mutation is a L to I mutation at position 53, 56, 80, or 118.
  • 89CC The therapeutic compound of any of embodiments 89H-89BB, wherein the IL-2 mutein further comprises a mutation at one or more positions of 29, 31, 35, 37, 48, 69, 71, 74, 88, and 125 corresponding to those positions in SEQ ID NO: 6.
  • 89DD The therapeutic compound of any of embodiments 89H-89CC, wherein the IL-2 mutein further comprises a mutation at one or more of positions E15, H16, Q22, D84, E95, or Q126 or 1, 2, 3, 4, 5, or each of positions E15, H16, Q22, D84, E95, or Q126 is wild-type.
  • 89EE The therapeutic compound of any of embodiments 89H-89DD, wherein the mutation in the mutein is one or more of E15Q, H16N, Q22E, D84N, E95Q, or Q126E.
  • 89FF The therapeutic compound of any of embodiments 89H-89EE, wherein the mutein comprises a N29S mutation in SEQ ID NO: 6.
  • 89GG The therapeutic compound of any of embodiments 89H-89FF, wherein the mutein comprises a Y31S or a Y51H mutation.
  • 89HH The therapeutic compound of any of embodiments 89H-89GG, wherein the mutein comprises a K35R mutation.
  • 89JJ The therapeutic compound of any of embodiments 89H-89II, wherein the mutein comprises a K48E mutation.
  • 89KK The therapeutic compound of any of embodiments 89H-89JJ, wherein the mutein comprises a V69A mutation.
  • 89LL The therapeutic compound of any of embodiments 89H-89KK, wherein the mutein comprises a N71R mutation.
  • 89MM The therapeutic compound of any of embodiments 89H-89LL, wherein the mutein comprises a Q74P mutation.
  • 89NN The therapeutic compound of any of embodiments 89H-89MM, wherein the mutein comprises a N88D or a N88R mutation.
  • 89PP The therapeutic compound of any of embodiments 89H-8900, wherein the IL-2 mutein is fused or linked to a Fc peptide.
  • the therapeutic compound of embodiment 89PP1, wherein the Fc peptide comprises L247A, L248A, and/or a G250A mutations (Rabat numbering).
  • the therapeutic compound of embodiment 89PP1, wherein the Fc peptide comprises a L234A mutation, a L235A mutation, and/or a G237A mutation (EU numbering).
  • the therapeutic compound of embodiment 89H wherein the compound comprises a polypeptide comprising a first chain and a second chain that form the polypeptide, wherein
  • the first chain comprises:
  • V H is a variable heavy domain that binds to the target cell with a V L domain of the second chain
  • H c is a heavy chain of antibody comprising CH1-CH2-CH3 domain
  • the Linker is a gly cine/ serine linker
  • Ci is a IL-2 mutein fused or linked to a Fc protein in either the N-terminal or C-terminal orientation
  • the second chain comprises:
  • V L is a variable light chain domain that binds to the target cell with the V H domain of the first chain
  • the Lc domain is a light chain CK domain
  • the therapeutic compound of embodiment 89QQ wherein the VH and VL domain are anti-MAdCAM variable domains that bind to MAdCAM expressed on a cell.
  • 89QQ2 The therapeutic compound of embodiment 89QQ or 89QQ1, wherein the IL-2 mutein comprises a mutation at a position that corresponds to position 53, 56, 80, or 118 of SEQ ID NO: 6.
  • 89QQ4 The therapeutic compound of embodiments 89QQ2 or 89QQ3, wherein the mutein further comprises a mutation at a position that corresponds to position 69, 75, 88, and/or 125, or any combination thereof.
  • 89QQ5. The therapeutic compound of embodiments 89QQ2 or 89QQ3, wherein the IL-2 mutein comprises a mutation selected from the group consisting of: at one of L53I, L56I, L80I, and Ll 181 and the mutations of V69A, Q74P, N88D or N88R, and optionally C125A or C125S. 89QQ6.
  • 89QQ11 The therapeutic compound of any one of embodiments 89QQ-89QQ10, wherein the Fc protein comprises L247A, L248A, and G250A mutations or a L234A mutation, a L235A mutation, and/or a G237A mutation according to KABAT numbering.
  • 89QQ12 The therapeutic compound of any one of embodiments 89QQ-89QQ11, wherein the Linker comprises a sequence of GGGGSGGGGSGGGGS or
  • 89QQ13 The therapeutic compound of any one of embodiments 89QQ-89QQ11, wherein the polypeptide comprises a Fc peptide comprising a sequence described herein.
  • Rl, R2, R3 and R4 comprises an anti -BCR antibody molecule, e.g., an antagonistic anti- BCR antibody molecule, one comprises an anti FCRL antibody molecule, and one comprises specific targeting moiety.
  • the anti-FCRL molecule comprises: an anti-FCRL antibody molecule, e.g., an agonistic anti- FCRL antibody molecule, directed to FCRL1, FCRL2, FCRL3, FCRL4, FCRL5, or FCRL6.
  • an anti-FCRL antibody molecule e.g., an agonistic anti- FCRL antibody molecule, directed to FCRL1, FCRL2, FCRL3, FCRL4, FCRL5, or FCRL6.
  • Rl, R2, R3 and R4 each independently comprise:
  • an effector binding/modulating moiety e.g., an ICIM binding/modulating moiety, an IIC binding/modulating moiety, or an SM binding/modulating moiety, that minimizes or inhibits T cell activity, expansion, or function (a T cell effector binding/modulating moiety);
  • an effector binding/modulating moiety e.g., an ICIM binding/modulating moiety, an IIC binding/modulating moiety, or an SM binding/modulating moiety, that minimizes or inhibits B cell activity, expansion, or function (a B cell effector binding/modulating moiety);
  • iv) is absent; provided that, a T cell effector binding/modulating moiety, a B cell effector binding/modulating moiety, and a specific targeting moiety are present.
  • one of Rl, R2, R3, and R4 comprises an agonistic anti-PD-l antibody and one comprises an HLA-G molecule.
  • Rl, R2, R3, and R4 comprises an SM binding/modulating moiety, e.g., a CD39 molecule or a CD73 molecule.
  • Rl, R2, R3, and R4 comprises an entity that binds, activates, or maintains, a regulatory immune cell, e.g., a Treg cell or a Breg cell.
  • Rl, R2, R3, and R4 comprises an agonistic anti-PD-l antibody or one comprises an HLA- G molecule.
  • one of Rl, R2, R3, and R4 comprises an agonistic anti-PD-l antibody, one comprises an HLA-G molecule, and one comprises CD39 molecule or a CD73 molecule.
  • binding/modulating moiety comprises a polypeptide
  • binding/modulating moiety comprises a polypeptide having at least 5, 10, 20, 30, 40, 50, 150,
  • binding/modulating moiety has a molecular weight of 5, 10, 15, 20, or 40 Kd.
  • therapeutic compound of any of embodiments 1-101 wherein the therapeutic compound does not specficially target a tissue selected from lung, skin, pancreas, retina, prostate, ovary, lymph node, adrenal gland, liver or gut tissue.
  • 105 The therapeutic compound of any of embodiments 1-101, wherein the therapeutic compound does not specficially target TIE-2, APN, TEM4, TEM6, ICAM-l, nucleolin P2Z receptor, Trk-A, FLJ10849, HSPA12B, APP, or OX-45.
  • 106 The therapeutic compound of any of embodiments 1-101, wherein the therapeutic compound does not specficially target a luminally expressed protein.

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