HK40015990A - Targeted immunotolerance - Google Patents

Description

Targeted immune tolerance

Cross Reference to Related Applications

This application claims priority to U.S. provisional application No. 62/471,509, filed on 2017, 3, 15, which is incorporated herein by reference in its entirety.

Technical Field

Embodiments provided herein relate to methods and compositions, e.g., for local or targeted immune-privileged.

Background

Examples of undesired immune responses, such as rejection of transplanted tissue or autoimmune disorders, for example, constitute a major health problem for millions of people worldwide. The long-term outcome of organ transplantation is often characterized by chronic rejection and eventual failure of the transplanted organ. Over twenty autoimmune disorders are known, which affect essentially every organ of the body, and affect more than five thousand people in north america alone. Broadly effective immunosuppressive drugs used to combat pathogenic immune responses in both cases have serious side effects.

Disclosure of Invention

Disclosed herein are methods and therapeutic compounds that provide site-specific immune-privileged activity. The embodiments disclosed herein are incorporated by reference in this section.

In some embodiments, the therapeutic compound comprises an engineered multispecific compound, e.g., an engineered bispecific molecule, e.g., an engineered bispecific antibody molecule, comprising:

1) a specific targeting moiety selected from the group consisting of:

a) donor-specific targeting moieties that, for example, preferentially bind to a donor target (e.g., preferentially bind to an acceptor antigen) and are suitable for providing site-specific immune-privileged for a transplanted tissue, e.g., an organ, from a donor; or

b) A tissue-specific targeting moiety that, e.g., preferentially binds to a target tissue of a subject (e.g., preferentially over a non-target tissue of a subject), and is suitable for providing site-specific immune-privileged for a tissue of a subject experiencing an undesirable immune attack (e.g., an autoimmune disorder); and

2) an effector binding/modulating moiety selected from the group consisting of:

(a) an immune cell inhibitory molecule binding/modulating moiety (referred to herein as an ICIM binding/modulating moiety);

(b) an immunosuppressive immune cell binding/modulating moiety (referred to herein as an IIC binding/modulating moiety); or

(c) The following effector binding/modulating moieties: it promotes an immunosuppressive local microenvironment as part of a therapeutic compound, for example by providing a substance proximal to the target that inhibits or minimizes the attack of the target's immune system (referred to herein as an SM binding/modulating moiety).

Effector binding/modulating moieties may fall into more than one of classes a, b and c. For example, as shown below, CTLA 4-binding molecules fall into both classes a and b.

In some embodiments, a therapeutic compound comprises an ICIM binding/modulating moiety. In some embodiments, the ICIM binding/modulating molecule binds to and agonizes an inhibitory molecule, such as an inhibitory immune checkpoint molecule, or otherwise inhibits or reduces the activity of an immune cell, such as a cytotoxic T cell, B cell, NK cell, or myeloid cell, such as a neutrophil or macrophage.

In some embodiments, the therapeutic compound comprises an engineered multispecific compound, e.g., an engineered bispecific molecule, e.g., an engineered bispecific antibody molecule, comprising:

1) a specific targeting moiety, such as a donor-specific targeting moiety (which binds to a donor target and is suitable for providing site-specific immune-privileged tissue from a donor, such as an organ) or a tissue-specific targeting moiety (which binds to a subject tissue target and is suitable for providing site-specific immune-privileged tissue from a subject undergoing an undesirable immune attack, such as an autoimmune disorder); and

2) an effector binding/modulating moiety comprising an ICIM binding/modulating moiety that binds to an effector molecule (e.g., an inhibitory receptor, such as PD-1) on an immune cell, wherein upon binding of a specific targeting moiety to its target and binding of the ICIM binding/modulating moiety to the effector molecule on the immune cell, the activity of the immune cell, e.g., the ability of the immune cell to increase immune attack, is down-regulated according to clustering of the effector molecule on the immune cell (e.g., by an inhibitory signal). In some embodiments, the engineered multispecific compound comprises an additional binding moiety such that the compound binds more than two specific molecules, such as, but not limited to, 3 or 4.

In some embodiments, a therapeutic compound comprises an ICIM binding/modulating moiety and has one or both of the following properties: (a) the level of down-regulation of immune cells when the therapeutic compound binds to its target is greater than the level of down-regulation of immune cells when the therapeutic compound does not bind to its target; and (b) the therapeutic compound, when conjugated to a cell surface inhibitory receptor (e.g., PD-1) on an immune cell, does not inhibit or does not significantly inhibit the ability of the cell surface inhibitory receptor to bind an endogenous ligand.

In some embodiments, the level of downregulation of immune cells when the therapeutic compound is bound to its target is greater than the level of downregulation of immune cells when the therapeutic compound is not bound to its target. In embodiments, the downregulation level of a therapeutic compound that binds to a target is equal to or 1.5-fold, 2-fold, 4-fold, 8-fold, or 10-fold greater than the downregulation level seen when the therapeutic compound is not bound to its target. In embodiments, the therapeutic compound does not down-regulate or does not significantly down-regulate immune cells when not bound to the target. Thus, indiscriminate or unwanted agonism of inhibitory receptors (e.g., PD-1) is minimized or eliminated. For example, when a therapeutic compound is bound to an immune cell but not to a targeting moiety, engagement of the inhibitory immune checkpoint molecule with the therapeutic compound does not result in downregulation or does not result in significant downregulation, e.g., inhibitory receptors on the immune cell that bind to the therapeutic compound do not cluster or cluster sufficiently to generate an inhibitory signal sufficient to cause downregulation or significant inhibition of the immune cell.

In embodiments, the therapeutic compound, when conjugated to a cell surface inhibitory receptor (e.g., PD-1) on an immune cell, does not inhibit or does not significantly inhibit the ability of the cell surface inhibitory receptor to bind an endogenous ligand. In some embodiments, the therapeutic compound binds to the PD-L1/2 binding site on PD-1. Thus, indiscriminate or unwanted antagonism of inhibitory receptors (e.g., PD-1) is minimized or eliminated. In embodiments, binding of the therapeutic compound to an inhibitory receptor (e.g., PD-1) on an immune cell does not interfere, or does not significantly interfere, with the ability of the inhibitory receptor to bind a natural ligand (e.g., PD-L1). In embodiments, binding of the therapeutic compound to an immunosuppressive receptor (e.g., PD-1) reduces binding of the natural ligand (e.g., PD-1) by less than 50, 40, 30, 20, 10, or 5% of the binding seen in the absence of the therapeutic compound.

In some embodiments, a therapeutic compound comprises an ICIM binding/modulating moiety and, when administered to a subject in a therapeutically effective dose, does not result in unacceptable levels of systemic immunosuppression, which may occur when agonism of inhibitory receptors in all types of immune cells (e.g., all T cells) occurs; or does not result in unacceptable levels of systemic immune activation, which may occur when a therapeutic compound antagonizes the interaction of the inhibitory receptor with the natural ligand.

While not wishing to be bound by theory, it is believed that upon administration to a subject, a therapeutic compound comprising an ICIM binding/modulating moiety may be present in any one of four states: i) unbound and in free solution form; ii) binds to inhibitory receptors expressed on the surface of immune cells (e.g., T cells) only through the ICIM binding/modulating moiety; iii) binding to the surface of the target graft or subject tissue only through the targeting moiety; iv) binding to the surface of a target graft or tissue of a subject via a targeting moiety and binding to an inhibitory receptor expressed by an immune cell (e.g., T cell) via an ICIM binding/modulating moiety. When therapeutic compound (iii) binds to the superficial target graft or subject tissue only through the targeting moiety, it has no or no significant effect on the target graft or tissue. When a therapeutic compound binds to a target graft or tissue through a targeting moiety and to an inhibitory receptor expressed by immune cells (e.g., T cells) through an ICIM binding/modulating moiety (iv), it produces immune-privileged responses at the target organ or tissue. While not wishing to be bound by theory, it is believed that this is achieved by multimerizing the therapeutic compound molecules on its surface by the target graft or donor tissue, for example, by immobilizing multiple therapeutic compound molecules at high density and valency. Multimerization of the therapeutic compound molecules allows the ICIM binding/modulating portion of the therapeutic compound to facilitate clustering of inhibitory receptors (e.g., pathogenic T cells) expressed on the surface of immune cells, as well as for silencing or down-regulating the transmission of inhibitory signals by immune cells. For example, in the case of T cells, a therapeutic compound comprising an ICIM binding/modulating moiety comprising a PD-L1 molecule or an anti-PD-1 Ab may be used. Binding of multiple therapeutic compound molecules to the target results in multimerization of the therapeutic compound molecules, which in turn results in clustering of PD-1 on T cells via the PD-L1 molecule or a functional anti-PD-1 antibody molecule. If this clustering occurs in the context of target MHC presentation to T cell receptor antigen on T cells, a negative signal is generated and the T cells will be inactivated. In embodiments, an ICIM binding/modulating moiety, such as a functional antibody molecule, binds to an effector molecule but does not inhibit or does not substantially inhibit the interaction of the effector molecule with its natural ligand.

In some embodiments, the therapeutic compound comprises an IIC binding/modulating moiety that binds and recruits immunosuppressive immune cells (e.g., tregs, such as Foxp3+ CD25+ tregs) to the vicinity of the target tissue.

In some embodiments, the therapeutic compound comprises an SM binding/modulating moiety that modulates, e.g., binds and inhibits, sequesters, degrades, or otherwise neutralizes a substance, e.g., a soluble molecule that modulates an immune response, such as ATP or AMP.

In some embodiments, the therapeutic compound comprises a targeting moiety specific for a target on an immune cell. In some embodiments, the target is as described herein. In some embodiments, the target is MAdCAM. In some embodiments, the targeting moiety is an antibody that binds to MAdCAM.

In some embodiments, the therapeutic compound comprises a donor-specific targeting moiety and provides site-specific immune-exemption to donor transplant tissue implanted in the subject. In some embodiments, the therapeutic compound comprises a tissue-specific targeting moiety and provides site-specific immune-privileged immunity to a tissue of the subject (e.g., a tissue afflicted with an undesired immune response in an autoimmune disorder).

The targeting moiety is specific to the donor graft or tissue of the subject protected by the immune system. In some embodiments, the effector molecule binding moiety comprises a de novo generated binding domain, such as a functional antibody molecule. In some embodiments, the effector binding/modulating moiety comprises an amino acid sequence derived from a natural ligand that recognizes an inhibitory receptor expressed on the surface of an immune cell (e.g., a T cell).

In some embodiments, the therapeutic compound silences immune cells (e.g., T cells) in proximity to the graft or donor tissue to be protected, but does not silence immune cells (e.g., T cells) not in proximity to the target, as the therapeutic compound requires the functional presence of the target graft or donor tissue. This is in contrast to therapeutic compounds which bind only to inhibitory receptors expressed by immune cells (e.g., T cells), in which case there are no functional consequences.

The methods and therapeutic compounds described herein are based, at least in part, on providing site-specific immune-sparing. The therapeutic compounds described herein and methods of use thereof allow for the minimization, e.g., reduction or elimination, of non-site-specific systemic administration of immunosuppressive therapeutics in a clinical setting, e.g., a setting in which it is desirable to reverse and suppress an immune response, e.g., in an autoimmune disease or tissue (e.g., organ, transplant). While clinically meaningful responses can result when the underlying pathophysiology driven by the aberrant immune system is affected, broadly effective immunosuppressive agents have the undesirable effect of reducing the patient's systemic immune system function. Patients undergoing chronic immunosuppression are at increased risk for infection and cancer, as the normally functioning immune system functions to fight the ongoing attack of pathogenic and opportunistic organisms present in the surrounding environment and to continually clear cancer cells from healthy individuals. The methods and therapeutic compounds described herein provide the following therapies: it selectively targets and attenuates, reduces or eliminates only the pathogenic immune response at the pathological site with minimal suppression of normal systemic immune system function elsewhere.

In some embodiments, a therapeutic compound as provided herein is provided. In some embodiments, the compound comprises i) a specific targeting moiety selected from the group consisting of: a) a donor-specific targeting moiety that, for example, preferentially binds to a donor target; or b) a tissue-specific targeting moiety that, e.g., preferentially binds to a target tissue of a subject; and ii) an effector binding/modulating moiety selected from: (a) an immune cell inhibitory molecule binding/modulating moiety (ICIM binding/modulating moiety); (b) an immunosuppressive immune cell binding/modulating moiety (IIC binding/modulating moiety); or (c) an effector binding/modulating moiety that promotes an immunosuppressive local microenvironment as part of a therapeutic compound, for example by providing a substance proximal to the target that inhibits or minimizes the attack of the target's immune system (SM binding/modulating moiety).

In some embodiments, 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 anti-ligand molecule comprises a PD-L1 molecule. In some embodiments, the ICIM is wherein the inhibitory immune molecule anti-ligand molecule is conjugated to a cognate inhibitory immune checkpoint molecule selected from PD-1, KIR2DL4, LILRB1, LILRB, or CTLA-4. In some embodiments, the ICIM is an antibody. In some embodiments, the ICIM comprises an antibody that binds to PD-1, KIR2DL4, LILRB1, LILRB, or CTLA-4. In some embodiments, the ICIM binding/modulating moiety comprises a functional antibody molecule directed against a cell surface inhibitory molecule.

In some embodiments, the cell surface inhibitory molecule is an inhibitory immune checkpoint molecule. In some embodiments, the inhibitory immune checkpoint molecule is selected from PD-1, KIR2DL4, LILRB1, LILRB2, CTLA-4, or from table 1.

In some embodiments, the effector binding/modulating moiety comprises an IIC binding/modulating moiety.

In some embodiments, the compound has the formula from N-terminus to C-terminus:

r1- -connecting sub-region A- -R2 or R3- -connecting sub-region B- -R4,

wherein R1, R2, R3 and R4 each independently comprise an effector binding/modulating moiety, such as 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.

In some embodiments, provided herein are methods of treating an autoimmune disorder or condition comprising administering one or more therapeutic compounds provided herein.

In some embodiments, provided herein are methods of treating a disease or condition comprising administering one or more therapeutic compounds provided herein.

In some embodiments, provided are methods of treating a subject having an inflammatory bowel disease, comprising administering to the subject a therapeutic compound provided herein to treat the inflammatory bowel disease. In some embodiments, the subject has Crohn's disease and or ulcerative colitis.

In some embodiments, there is provided a method of treating a subject having autoimmune hepatitis, the method comprising administering to the subject a therapeutic compound as provided herein to treat the autoimmune hepatitis.

In some embodiments, there is provided a method of treating primary sclerosing cholangitis, the method comprising administering to a subject a therapeutic compound as provided herein to treat the primary sclerosing cholangitis.

In some embodiments, there is provided a method of treating type 1 diabetes, the method comprising administering to a subject a therapeutic compound as provided herein to treat the type 1 diabetes.

In some embodiments, there is provided a method of treating a transplant subject, the method comprising administering to the subject a therapeutically effective amount of a therapeutic compound as provided herein, thereby treating the transplant (recipient) subject.

In some embodiments, there is provided a method of treating GVHD in a subject having transplanted donor tissue, the method comprising administering to the subject a therapeutically effective amount of a therapeutic compound as provided herein.

In some embodiments, there is provided a method of treating a subject having, at risk of having, or at increased risk of having an autoimmune disorder, the method comprising administering a therapeutically effective amount of a therapeutic compound, thereby treating the subject.

Drawings

Figure 1 is a depiction of a bispecific therapeutic compound in tandem scFv-Fc format (150kDa) containing a targeting scFv domain and an effector domain consisting of either a scFv or a sequence corresponding to an endogenous ligand.

Figure 2 is a depiction of T cells in combination with a therapeutic compound disclosed herein. In state 1, the bispecific effector domain binds to the inhibitory receptor of T cells in the systemic circulation, with neither agonism nor antagonism of the receptor. In state 2, the bispecific targeting domain binds to the target organ, resulting in bispecific multimerization on the surface of the target organ. During T cell recognition of the target organ, the multimerized effector domain binds, aggregates and transmits a signal through the T cell inhibitory molecule.

Figure 1 depicts a non-limiting embodiment of a therapeutic compound provided herein.

Figure 2 depicts a non-limiting illustration of how the therapeutic compounds provided herein function.

Figure 3 depicts a non-limiting illustration of a therapeutic compound provided herein.

Figure 3A depicts a non-limiting illustration of a therapeutic compound provided herein.

Figure 4 depicts a non-limiting illustration of a therapeutic compound provided herein.

Figure 5 depicts a non-limiting illustration of a therapeutic compound provided herein.

Fig. 6 depicts a non-limiting illustration of a therapeutic compound provided herein.

Figure 7 depicts a non-limiting illustration of a therapeutic compound provided herein.

Fig. 8 depicts a non-limiting illustration of a therapeutic compound provided herein.

Figure 9 depicts a non-limiting illustration of a therapeutic compound provided herein.

Figure 10 depicts a non-limiting illustration of a therapeutic compound provided herein.

Fig. 11 depicts a non-limiting illustration of a therapeutic compound provided herein.

Figure 12 depicts a non-limiting illustration of a therapeutic compound provided herein.

Figure 13 depicts a non-limiting illustration of a therapeutic compound provided herein.

Figure 14 depicts a non-limiting illustration of a therapeutic compound provided herein.

Figure 15 depicts a non-limiting illustration of a therapeutic compound provided herein.

Figure 16 depicts a non-limiting illustration of a therapeutic compound provided herein.

Figure 17 depicts a non-limiting illustration of a therapeutic compound provided herein.

Figure 18 depicts a non-limiting illustration of a therapeutic compound provided herein.

Detailed Description

As used herein and unless otherwise specified, the term "about" means ± 5% of its modified value. Thus, about 100 means 95 to 105.

As used herein and in the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

As used herein, the term "about" means that the numerical values are approximations, and that small changes will not significantly affect the practice of the disclosed embodiments. Where numerical limitations are used, "about" means that the numerical values can vary by ± 10% and still be within the scope of the disclosed embodiments unless the context indicates otherwise.

As used herein, the term "animal" includes, but is not limited to, humans and non-human vertebrates, such as wild animals, domestic animals and farm animals.

As used herein, the term "contacting" means binding two elements together in an in vitro system or in an in vivo system. For example, "contacting" a therapeutic compound with a subject or patient or cell includes administering the compound to the subject or patient (such as a human), as well as, for example, introducing the compound into a sample containing a target comprising a cell or purified preparation.

As used herein, the term "comprising" (and any form of comprising, such as "comprises", "comprises" and "comprising)", "having" (and any form of having, such as "having" and "has)", "including" (and any form of including, such as "includes" and "includes)", or "containing" (and any form of containing, such as "containing" and "containing)" are inclusive or open-ended and do not exclude additional, undescribed elements or method steps. Any composition or method that references the term "comprising" should also be understood to also describe such composition as constituting, consisting of, or consisting essentially of the recited components or elements.

As used herein, the terms "individual," "subject," or "patient," used interchangeably, mean any animal, including mammals, such as mice, rats, other rodents, rabbits, dogs, cats, pigs, cows, sheep, horses, or primates, such as humans.

As used herein, the term "inhibit" refers to a decrease in the result, symptom, or activity as compared to the activity or result in the absence of a compound that inhibits the result, symptom, or activity. In some embodiments, the result, symptom, or activity is inhibited by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%. The result, symptom or activity is also inhibited when completely eliminated or cleared.

As used herein, the phrase "in need thereof" refers to a subject that has been identified as in need of a particular method or treatment. In some embodiments, the identification may be by any diagnostic means. In any of the methods and treatments described herein, the subject may be in need of such methods and treatments. In some embodiments, the subject is in or will travel to an environment in which a particular disease, disorder, or condition is prevalent.

As used herein, the phrase "integer from X to Y" means any integer including the endpoints. For example, the phrase "an integer from X to Y" means 1, 2, 3, 4, or 5.

As used herein, the term "mammal" means a rodent (i.e., mouse, rat, or guinea pig), monkey, cat, dog, cow, horse, pig, or human. In some embodiments, the mammal is a human.

As used herein, the phrase "ophthalmically acceptable" refers to no sustained detrimental effect on the treated eye or its function or on the overall health of the treated subject. However, it will be appreciated that transient effects such as mild irritation or "stinging" sensations are common for topical ophthalmic administration, and that the presence of such transient effects is consistent with the definition of a composition, formulation or ingredient (e.g., excipient) herein as being "ophthalmically acceptable". In some embodiments, the pharmaceutical composition may be ophthalmically acceptable or suitable for ophthalmic administration.

By "specifically binds" or "specifically binds to" or is "specific for" a particular antigen, target or epitope, it is meant that the binding is measurably different from the non-specific interaction. Specific binding can be measured, for example, by determining the binding of the molecule compared to the binding of a control molecule, which is typically a similarly structured molecule 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 to a particular antigen, target or epitope can be at least about 10 for an antigen or epitope, e.g., by an antibody^-4MAt least about 10^-5MAt least about 10^-6MAt least about 10^-7MAt least about 10^-8MAt least about 10^-9MOr at least about 10^-10MAt least about 10^-11MAt least about 10^-12MOr greater K_DIn which K is_DRefers to the off-rate of a particular antibody-target interaction. In generalK of antibody specifically binding to antigen or target relative to antigen or epitope_DLarger, or at least 2-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 500-fold, 1000-fold, 5,000-fold, 10,000-fold, or more than a control molecule.

In some embodiments, specific binding to a particular antigen, target, or epitope can be, for example, by antibody to the target, antigen, or epitope K_AOr K_aK to target, antigen or epitope in comparison to control_AOr K_aAt least 2-fold, 4-fold, 5-fold, 20-fold, 50-fold, 100-fold, 500-fold, 1000-fold, 5,000-fold, 10,000-fold or more greater, wherein K is present_AOr K_aRefers to the binding rate of a particular antibody-target interaction.

As provided herein, therapeutic compounds and compositions may be used in the methods of treatment provided herein. As used herein, the terms "treatment", "treating" or "treating" mean both therapeutic treatment and prophylactic measure, wherein the aim is to alleviate an undesired physiological condition, disorder or disease, or to obtain a beneficial or desired clinical effect. For the purposes of these embodiments, beneficial or desired clinical effects include, but are not limited to, reduction of symptoms; reducing the extent of a condition, disorder or disease; a stable (i.e., not worsening) condition, disorder or disease state; delaying the onset of or slowing the progression of the condition, disorder or disease; ameliorating a condition, disorder or disease state or remission (whether partial or total), whether detectable or undetectable; improving at least one measurable physical parameter, not necessarily discernible by the patient; or promoting or ameliorating a condition, disorder or disease. Treatment includes eliciting a clinically significant response without excessive side effects. Treatment also includes an extended survival compared to the expected survival when not receiving treatment. Thus, "pain treatment" or "treating pain" means an activity that reduces or ameliorates any primary phenomenon or secondary symptom associated with pain or other condition described herein.

Provided herein are therapeutic compounds, e.g., therapeutic protein molecules, e.g., fusion proteins, that include a targeting moiety and an effector binding/modulating moiety, typically in the form of separate domains. Methods of using and making the therapeutic compounds are also provided. The targeting moiety is used to localize the therapeutic compound and hence the effector binding/modulating moiety to the site where immune-privileged immunity is desired. The effector binding/modulating moiety comprises one or more of: (a) an immune cell inhibitory molecule binding/modulating moiety (ICIM binding/modulating moiety); (b) an immunosuppressive immune cell binding/modulating moiety (IIC binding/modulating moiety); or (c) a soluble molecule binding/modulating moiety (SM binding/modulating moiety). In some embodiments, the therapeutic compound comprises: (a) and (b); (a) and (c); (b) and (c); or (a), (b) and (c).

The present disclosure provides molecules that can act, for example, as PD-1 agonists. Without being bound by any particular theory, agonism of PD-1 inhibits T cell activation/signaling and may be achieved by different mechanisms. For example, it has been described that cross-linking can result in agonistic, bead-bound functional PD-1 agonists (Akkaya. Ph.D. topic: Modulation of the PD-1 pathway by inhibition antibody superagonists. Christ Church College, Oxford, UK,2012), which is incorporated herein by reference. Crosslinking of PD-1 with two mabs that bind non-overlapping epitopes induces PD-1 signaling (Davis, US 2011/0171220), which is incorporated herein by reference. Another example is illustrated by the use of goat anti-PD-1 antisera (e.g., AF1086, R & D Systems), which are incorporated herein by reference, which act as agonists when soluble (Said et al, 2010, Nat Med), which are incorporated herein by reference. Non-limiting examples of PD-1 agonists that can be used in embodiments of the invention 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 (Anapys/cell), PD1-17, PD1-28, PD1-33 and PD1-35(Collins et al, US 2008/0311117A 1 Antibodies against PD-1 and uses therefor, which is incorporated herein by reference), or may be a bispecific monovalent anti-PD-1/anti-CD 3(Ono) and the like. In some embodiments, the PD-1 agonist antibody can be an antibody that blocks the binding of PD-L1 to PD-1. In some embodiments, the PD-1 agonist antibody can be an antibody that does not block the binding of PD-L1 to PD-1.

PD-1 agonism may be measured by any method, such as the methods described in the examples. For example, cells can be constructed that express (including stably express) constructs that include a human PD-1 polypeptide "enzyme donor" fused to b-galactosidase and 2) an SHP-2 polypeptide "enzyme receptor" fused to b-galactosidase. Without being bound by any theory, when PD-1 is engaged, SHP-2 is recruited to PD-1. The enzyme acceptor and enzyme donor form a fully active b-galactosidase that can be measured. Although, this assay does not directly show PD-1 agonism, it shows activation of PD-1 signaling. PD-1 agonism may also be measured by measuring inhibition of T cell activation, as without being bound by any theory PD-1 agonism inhibits anti-CD 3-induced T cell activation. For example, PD-1 agonism can be measured by pre-activating T cells with PHA (for human T cells) or ConA (for mouse T cells) so that they express PD-1. Cells can then be reactivated with anti-CD 3 in the presence of anti-PD-1 (or PD-L1) for use in PD-1 agonism assays. T cells receiving PD-1 agonist signaling in the presence of anti-CD 3 will show reduced activation relative to anti-CD 3 stimulation alone. Activation can be read by proliferation or cytokine production (IL-2, IFNg, IL-17) or other markers such as the CD69 activation marker. Thus, PD-1 agonism can be measured by cytokine production or cell proliferation. Other methods can also be used to measure PD-1 agonism.

PD-1 is a member of the Ig superfamily expressed on activated T cells and other immune cells. The natural ligands for PD-1 appear to be PD-L1 and PD-L2. Without being bound by any particular theory, when PD-L1 or PD-L2 binds to PD-1 on activated T cells, an inhibitory signaling cascade is initiated, resulting in a diminished function of the activated T effector cells. Thus, blocking the interaction between PD-1 on a T cell and PD-L1/2 on another cell (e.g., a tumor cell) with a PD-1 antagonist is referred to as checkpoint inhibition, and the T cell is released from the inhibition. In contrast, PD-1 agonist antibodies can bind to PD-1 and send inhibitory signals and attenuate T cell function. Thus, PD-1 agonist antibodies can be incorporated as effector molecule binding/modulating moieties in the various embodiments described herein, which can effect local tissue-specific immunomodulation when paired with targeting moieties.

The effector molecule binding/modulating moiety may provide an immunosuppressive signal or environment in a variety of ways. In some embodiments, the effector binding/modulating moiety comprises an ICIM binding/modulating moiety that directly binds to and (under appropriate conditions as described herein) activates an inhibitory receptor expressed by immune cells responsible for driving disease pathology. In another embodiment, the effector binding/modulating moiety comprises an IIC binding/modulating moiety that binds to and accumulates an immunosuppressive immune cell. In some embodiments, the accumulated immunosuppressive cells promote immune-privileged cells. In another embodiment, the effector binding/modulating moiety comprises an SM binding/modulating moiety that manipulates the surrounding microenvironment such that it is less permissive for the function of immune cells (e.g., immune cells that drive a disease condition). In some embodiments, the SM binding/modulating moiety depletes entities that facilitate immune attack or activation.

The targeting moiety and the effector binding/modulating moiety are physically tethered to each other, either directly or through a linking entity, covalently or non-covalently, for example as members of the same protein molecule in a therapeutic protein molecule. In some embodiments, the targeting and effector moieties are typically provided in separate domains in the therapeutic protein molecule (e.g., fusion protein). In some embodiments, the targeting moiety, the effector binding/modulating moiety, or both each comprise a single domain antibody molecule, for example a camelid antibody VHH molecule or a human soluble VH domain. It may also comprise a single chain fragment variable domain (scFv) or Fab domain. In some embodiments, a therapeutic protein molecule or a nucleic acid (e.g., mRNA or DNA) encoding a therapeutic protein molecule can be administered to a subject. In some embodiments, the targeting and effector molecule binding/modulating moieties are attached to a third entity, such as a carrier (e.g., a polymeric carrier), a dendrimer, or a particle (e.g., a nanoparticle). Therapeutic compounds may 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 than systemic immunosuppressive function. The target or site may comprise donor tissue or autologous tissue.

Provided herein are methods of providing site-specific immune-privileged therapy with a therapeutic compound disclosed herein to transplanted donor tissue, such as allograft tissue, e.g., as described herein, e.g., allograft liver, allograft kidney, allograft heart, allograft pancreas, allograft thymus or thymus tissue, allograft skin or allograft lung. In embodiments, the treatment minimizes rejection of the donor graft tissue; minimizing immune effector cell-mediated damage to donor transplant tissue; prolonged acceptance of donor graft tissue; or to extend the functional life of the donor graft tissue.

Also provided herein are methods of inhibiting Graft Versus Host Disease (GVHD) by minimizing the ability of donor immune cells (e.g., donor T cells) to mediate immune attack of recipient tissue with the therapeutic compounds disclosed herein.

Also provided herein are methods of treating (e.g., therapeutically or prophylactically treating (or preventing)) an autoimmune disorder or response in a subject by administering a therapeutic compound disclosed herein, e.g., to provide site-or tissue-specific modulation of the immune system. In some embodiments, the method provides tolerance to a tissue of the subject; minimizing rejection of the subject's tissue; minimizing immune effector cell-mediated damage to the subject's tissue; or prolonging the function of a tissue of the subject. In some embodiments, the therapeutic compound includes a targeting moiety that targets (e.g., specifically targets) a tissue that is under or at risk of autoimmune attack. Non-limiting exemplary tissues include, but are not limited to, pancreas, myelin, salivary glands, synovial cells, and muscle cells.

As used herein, the terms "treatment", "treating" or "treating" relate to therapeutic treatment, wherein the aim is to alleviate an undesired physiological condition, disorder or disease, or to obtain a beneficial or desired clinical effect. For example, beneficial or desired clinical effects include, but are not limited to, alleviation of symptoms; reducing the extent of a condition, disorder or disease; a stable (i.e., not worsening) condition, disorder or disease state; delaying the onset of or slowing the progression of the condition, disorder or disease; ameliorating a condition, disorder or disease state or remission (whether partial or total), whether detectable or undetectable; improving at least one measurable physical parameter, not necessarily discernible by the patient; or promoting or ameliorating a condition, disorder or disease. Treatment includes eliciting a clinically significant response without excessive side effects. Treatment also includes an extended survival compared to the expected survival when not receiving treatment. Thus, "autoimmune disease/disorder treatment" means an activity that alleviates or ameliorates any primary or secondary symptoms associated with the autoimmune disease/disorder treatment or other condition described herein. Provided herein are various diseases or conditions. Therapeutic treatments may also be administered prophylactically to prevent or alleviate a disease or condition prior to onset of the disease.

In some embodiments, administration of the therapeutic compound is initiated after the condition is evident. In some embodiments, administration of the therapeutic compound begins before the onset or complete onset of the condition. In some embodiments, administration of the therapeutic compound begins before the onset or complete onset of the disorder, e.g., in a subject with the disorder, a high risk subject, a subject with a biomarker at risk or present of the disorder, a subject with a family history of the disorder, or other indicators of the risk or absence of symptoms of the disorder. For example, in some embodiments, a subject having islet cell damage but not yet having diabetes is treated.

While not wishing to be bound by theory, it is believed that the targeting moiety functions to bind and accumulate the therapeutic agent to a target that is selectively expressed at an anatomical site where immune-privileged immunity is desired. In some embodiments, for example in the context of donor tissue transplantation, the target moiety binds to a target, e.g., an allele product, that is present in the donor tissue but not in the recipient. For the treatment of autoimmune disorders, the targeting moiety binds to a target that is preferentially expressed at anatomical sites where exemption is desired (e.g., in the pancreas). For treatment of GVHD, the targeting moiety targets host tissue and protects the host from attack by transplanted immune effector cells from the transplanted tissue.

In addition, while not wishing to be bound by theory, it is believed that the effector binding/modulating moiety serves to deliver an immunosuppressive signal or otherwise create an immune-privileged environment.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which these embodiments belong. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Headings, sub-headings or numbering or letter elements, such as (a), (b), (i), etc., are presented for ease of reading only. The use of headings or numbers or alphabetical elements in this document does not require that the steps or elements be performed in alphabetical order or that the steps or elements be necessarily separated from one another. Other features, objects, and advantages of the embodiments will be apparent from the description and drawings, and from the claims.

Additional definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the embodiments belong. In describing and claiming embodiments of the present invention, where a definition is provided, the following terms and terms otherwise referenced in this application will be used in accordance with their definition.

It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

As used herein, an antibody molecule refers to a polypeptide comprising at least one functional immunoglobulin variable domain sequence, such as an immunoglobulin chain or fragment thereof. Antibody molecules encompass antibodies (e.g., full length antibodies) and antibody fragments. In some embodiments, the antibody molecule comprises a full-length antibody or an antigen-binding or functional fragment of a full-length immunoglobulin chain. For example, a full-length antibody is an immunoglobulin (Ig) molecule (e.g., an IgG antibody) that occurs naturally or is formed by the process of recombination of normal immunoglobulin gene fragments. In embodiments, an antibody molecule refers to an immunologically active antigen-binding portion of an immunoglobulin molecule, such as an antibody fragment. Antibody fragments, e.g. functional fragments, comprise 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). Functional antibody fragments bind to the same antigen as the antigen recognized by the intact (e.g., full-length) antibody. The term "antibody fragment" or "functional fragment" also includes isolated fragments consisting of variable regions, such as "Fv" fragments consisting of the variable regions of the heavy and light chains, or recombinant single chain polypeptide molecules in which the light and heavy chain variable regions are linked by a peptide linker ("scFv proteins"). In some embodiments, an antibody fragment does not include portions of the antibody that do not contain antigen binding activity, such as an Fc fragment or a single amino acid residue. Exemplary antibody molecules include full-length antibodies and antibody fragments, such as dAb (domain antibody), single chain, Fab ', and F (ab')2 fragments, and single chain variable fragments (scFv).

The term "antibody molecule" also encompasses all or an antigen-binding fragment of a domain or single domain antibody, which may also be referred to as an "sdAb" or "VHH". The domain antibody comprises V_HOr V_LIt can be used as an independent antibody fragment. In addition, domain antibodies include heavy chain-only antibodies (hcabs). The domain antibody also included the CH2 domain of IgG as the basic scaffold into which the CDR loops were grafted. It can also be generally defined as a polypeptide or protein comprising an amino acid sequence consisting of four framework regions interspersed with three complementarity determining regions. This is expressed as FR1-CDR1-FR2-CDR2-FR3-CDR3-FR 4. sdabs can be produced in camelids such as llamas, but can also be produced synthetically using techniques well known in the art. Numbering of amino acid residues of an sdAb or polypeptide is according to Kabat et alGeneral numbering of VH domains. ("Sequence of proteins of immunological interest," US Public Health Services, NIH Bethesda, MD, Pub. No. 91, which is incorporated herein by reference). According to this numbering, FR1 of the sdAb comprises the amino acid residues at positions 1-30, CDR1 of the sdAb comprises the amino acid residues at positions 31-36, FR2 of the sdAb comprises the amino acids at positions 36-49, CDR2 of the sdAb comprises the amino acid residues at positions 50-65, FR3 of the sdAb comprises the amino acid residues at positions 66-94, CDR3 of the sdAb comprises the amino acid residues at positions 95-102, and FR4 of the sdAb comprises the amino acid residues at positions 103-113. Domain antibodies are also described in WO2004041862 and WO2016065323, each of which is incorporated herein by reference. The domain antibody may be a targeting moiety as described herein.

The antibody molecule 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 have a higher order of specificity (e.g., tetraspecific) and/or a higher order of valency than hexavalent. An antibody molecule may comprise a functional fragment of the variable region of the light chain and a functional fragment of the variable region of the heavy chain, or the heavy and light chains may be fused together to form a single polypeptide.

Examples of versions of 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 on symmetric or asymmetric Fc patterns.

For example, the figures illustrate non-limiting and varying symmetric homodimer processes. In some embodiments, the dimerization interface is centered around the human IgG1CH2-CH3 domain, which is dimerized by a contact interface spanning CH2/CH2 and CH3/CH 3. The resulting bispecific antibody shown has a total valency consisting of four binding units, two identical binding units at the N-terminus of each side of the dimer, and two identical units at the C-terminus of each side of the dimer. In each case, the binding unit at the N-terminus of the homodimer is different from the binding unit at the C-terminus of the homodimer. The use of this type of bivalent for inhibitory T cell receptors at either end of the molecule and the use of bivalent for tissue tethering antigens can be achieved at either end of the molecule.

For example, in FIG. 3, a non-limiting embodiment is shown. The N-terminus of the homodimer contains two identical Fab domains and consists of two identical light chains, which are separate polypeptides, interacting with the N-terminal VH-CH1 domain of each heavy chain via VH/VL interactions and Ckappa or Clamba interaction with CH 1.A natural disulfide bond exists between Ckappa or clamba and CH1, providing a covalent anchor between the light and heavy chains. At the c-terminus of the design are two identical scFv units, where (in this example) the c-terminus of the CH3 domain of Fc is followed by a flexible hydrophilic linker typically consisting of (but not limited to) serine, glycine, alanine and/or threonine residues, followed by a VH domain of each scFv unit, which is followed by a glycine/serine rich linker, followed by a VL domain. These tandem VH and VL domains associate to form a single chain fragment variable region (scFv) that is appended to the c-terminus of the Fc. Due to the homodimeric nature centered on Fc, two such units are present at the c-terminus of the molecule. The domain order of the scFv may be configured to be either VH-linker-VL or VL-linker-VH from N to C terminus.

A non-limiting example of a molecule with different binding regions at different termini is where one end is a PD-1 agonist and the antibody that provides the target specificity is an anti-MAdCAM-1 antibody. This may be as shown, for example, in fig. 3A, which shows molecules in different orientations.

In some embodiments, the MAdCAM antibody is a blocking or non-blocking antibody as described elsewhere herein. Without being bound by any theory, MAdCAM has been shown to interact with the head of integrin α 4 β 7 expressed on lymphocytes through multiple residues within its two Ig superfamily I-set domains, and the atomic level structural basis of this 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). It has been shown in great detail in structure, mechanism and function that in both the human (Chen J et al (2003) Nat Struct biol.10, 995-1001; de Chateau M et al (2001) biochemistry.40,13972-13979) and mouse (Day ES et al (2002) Cell Communneeds.9, 205-219; Hoshino H et al (2011) J Histochem Cytochem.59,572-583) molecular systems, any interaction of MAdCAM with α 4 β 7 depends on the presence of three bi-cationic binding sites in the integrin β 7 subunit I-like domain, and these metal binding sites can coordinate with Ca2+, Mn2+ and Mg2 +. In the presence of high Ca2+, in the presence or absence of Mg2+ or Mn2+, using cell adhesion assays, flow cytometry and/or flow cell assays, it was demonstrated that MAdCAM/α 4 β 7 interaction has lower functional affinity and allows rolling adhesion of lymphocytes, while at high Mg2+ or Mn2+ with low Ca2+ but activated integrins, MAdCAM/α 4 β 7 interaction has higher functional affinity and mediates robust lymphocyte adhesion (Chen J et al (2003). Nat Struct biol.10, 995-1001). Many research groups have demonstrated that the effect of anti-MAdCAM or anti- α 4 β 7 antibodies on MAdCAM interaction with α 4 β 7 can be monitored using various Cell: Cell, Cell: membrane preparations and/or Cell: protein based adhesion/interaction assays using FACS, Cell flow chamber based counting or IHC based readout enabling one to recognize blocking or non-blocking antibodies (Nakache, M et al (1989) nature.337, 179-181; Streeter, PR et al (1988) nature.331.41-46; Yang Y et al (1995) and scid im.42.235-247; Leung E et al (2004) im. Biol. 82.400-409; pulen N et al (2009) B J. pharmacol.157.281-293; Soler D et al (checol) jPharmacol Exp heat.330.875J-875J 15784; Qi. 15759) 3-287).

This has been exemplified in the context of a mouse system using the identification of anti-mouse MAdCAM antibodies, such as MECA-89 (non-blocking) and MECA-367 (blocking) (Nakache, M et al (1989) Nature.337, 179-181; street, PR et al (1988) Nature.331.41-46; Yang Y et al (1995) Scan and JImmunol.42.235-247). In the human system, antibodies that block the interaction of human MAdCAM with human α 4 β 7 have been identified, such as anti-human MAdCAM PF-00547659(Pullen N et al (2009). B J pharmacol.157.281-293) and anti-human α 4 β 7-dimensional polyclonal mab (vedolizumab) (Soler D et al (2009). J Pharmacol expther.330.864-875), as well as antibodies that do not block the interaction, such as anti-human MAdCAM clone 17F5(Soler D et al (2009). J Pharmacol Exp ther.330.864-875) and anti-human α 4 β 7 clone J19(Qi J et al (2012). J Biol m.287.49.15749-15759). Thus, the antibody may be blocking or non-blocking based on the desired effect. In some embodiments, the antibody is a non-blocking MAdCAM antibody. In some embodiments, the antibody is a blocking MAdCAM antibody. 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 is incorporated by reference in its entirety.

In another example, and as depicted in FIG. 4, the N-terminus of the homodimer contains two identical Fab domains, consisting of two identical light chains, which are separate polypeptides, that interact with the N-terminal VH-CH1 domain of each heavy chain by VH/VL interaction and Ckappa or Clamba interaction with CH 1.A natural disulfide bond exists between Ckappa or clamba and CH1, providing a covalent anchor between the light and heavy chains. At the c-terminus of the design are two identical VH units (although non-antibody moieties may also be substituted at this point or at any of the four terminal junctions/fusions), where (in this example) the c-terminus of the CH3 domain of Fc is followed by a flexible hydrophilic linker typically consisting of (but not limited to) serine, glycine, alanine and/or threonine residues, followed by a soluble independent VL domain based on the VH3 germline family. Due to the homodimeric nature centered on Fc, two such units are present at the c-terminus of the molecule.

In another non-limiting example, as shown in fig. 5, the N-terminus of the homodimer contains two identical Fab domains, consisting of two identical light chains, which differ from fig. 3 and 4, physically bound to the heavy chain at the N-terminus by a linker between the c-terminus of Ckappa or clamba and the N-terminus of VH. The linker may be 36-80 amino acids long and consists of serine, glycine, alanine and threonine residues. The physically bound n-terminal light chain interacts with the n-terminal VH-CH1 domain of each heavy chain through VH/VL interactions and Ckappa or Clambda interaction with CH 1. There is a natural disulfide bond between Ckappa or Clambda and CH1, providing additional stability between the light and heavy chains. At the c-terminus of the design are two identical Fab units, where (in this example) the c-terminus of the CH3 domain of Fc is followed by a flexible hydrophilic linker typically composed of (but not limited to) serine, glycine, alanine and/or threonine residues, followed by a CH1 domain, followed by a VH domain at the c-terminus. Light chains designed to pair with the c-terminal CH1/VH domain are expressed as separate polypeptides, unlike the N-terminal light chain, which binds to the N-terminal VH/CH1 domain as described. The C-terminal light chain forms an interface between VH/VL and Ckappa or Clambda and CH 1. The natural disulfide bond anchors the light chain to the heavy chain. In addition, any antibody moiety at any of the four points of attachment/fusion may be replaced by a non-antibody moiety, e.g., not comprising an effector binding/modulating portion of an antibody molecule.

Bispecific antibodies can also be asymmetric, as shown in the following non-limiting examples. Non-limiting examples are also shown in fig. 6, 7 and 8, which illustrate the asymmetric/heterodimer approach. In addition, in any of these versions, any antibody moiety at any of the four junctions/fusions may be replaced by a non-antibody moiety, e.g., not comprising an effector binding/modulating portion of an antibody molecule. In some embodiments, the dimerization interface is centered around the human IgG1CH2-CH3 domain, which is dimerized by a contact interface spanning CH2/CH2 and CH3/CH 3. However, to achieve heterodimerization rather than homodimerization of each heavy chain, mutations were introduced in each CH3 domain. Heterodimerization mutations include a T366W mutation (kabat) in one CH3 domain and T366S, L368A and Y407V (kabat) mutations in another CH3 domain. The heterodimeric interface can be further stabilized with de novo disulfide bonds by mutating the native residues to cysteine residues (such as S354 and Y349) on opposite sides of the CH3/CH3 interface. The resulting bispecific antibody displayed had a total valency consisting of four binding units. By this approach, the entire molecule can be designed to have dual specificity at only one end and mono-specificity at the other end (tri-specificity overall) or dual specificity at either end, with a total molecule specificity of 2 or 4. In the illustrative examples below, the C-terminus comprises two identical binding domains, which can, for example, provide a bivalent monospecificity for tissue tethering targets. At the N-terminus of all three illustrative examples, the two binding domains comprise different recognition elements/paratopes, and recognition of two different epitopes on the same effector moiety target can be achieved, or recognition of, for example, T cell inhibitory receptor and CD3 can be recognized. In some embodiments, the N-terminal binding moiety may be interchanged with other single polypeptide formats, such as scFv, single chain Fab, tandem scFv, e.g., VH or VHH domain antibody configurations. Other types of recognition elements, such as linear or cyclic peptides, may also be used.

An example of an asymmetric molecule is depicted in fig. 6. Referring to FIG. 6, the N-terminus of the molecule consists of a first light chain paired with a first heavy chain by VH/VL and Ckappa or Clamba/CH 1 interaction and a covalent tether consisting of a native heavy/light chain disulfide bond. On the other side of this heterodimeric molecule at the N-terminus is a second light chain and a second heavy chain, which are physically joined by a linker between the c-terminus of Ckappa or Clamba and the N-terminus of VH. The linker may be 36-80 amino acids long and consists of serine, glycine, alanine and threonine residues. The physically bound n-terminal light chain interacts with the n-terminal VH-CH1 domain of each heavy chain through VH/VL interactions and Ckappa or Clambda interaction with CH 1. There is a natural disulfide bond between Ckappa or Clambda and CH1, providing additional stability between the light and heavy chains. At the c-terminus of the molecule are two identical soluble VH3 germline family VH domains linked to the c-terminus of the CH3 domains of heavy chain 1 and heavy chain 2 by N-terminal glycine/serine/alanine/threonine based linkers.

In some embodiments, the asymmetric molecule can be illustrated as described in fig. 7. For example, the N-terminus of the molecule is composed of two different VH3 germline-based soluble VH domains linked to the human IgG1 hinge region by a glycine/serine/alanine/threonine-based linker. The VH domain linked to the first heavy chain is different from the VH domain linked to the second heavy chain. At the c-terminus of each heavy chain is an additional soluble VH domain based on the VH3 germline, which VH domain is identical on each of the two heavy chains. The heavy chains heterodimerize via the knob-entry-hole mutations previously described, which are present at the CH3 interface of the Fc module.

In some embodiments, the asymmetric molecule can be as illustrated in fig. 8. This example is similar to the molecule shown in fig. 7, but the two N-terminal Fab units are configured in the following manner: light chain 1 and light chain 2 are physically associated with heavy chain 1 and heavy chain 2 through a linker between the c-terminus of Ckappa or Clambda and the N-terminus of each respective VH. The linkers can be in each case 36 to 80 amino acids long and consist of serine, glycine, alanine and threonine residues. The physically bound n-terminal light chain interacts with the n-terminal VH-CH1 domain of each heavy chain through VH/VL interactions and Ckappa or Clambda interaction with CH 1. There is a natural disulfide bond between Ckappa or Clambda and CH1, providing additional stability between the light and heavy chains.

Bispecific molecules may also have mixed versions. This is illustrated, for example, in fig. 9, 10 and 11.

For example, as illustrated in fig. 9, a homodimer Fc-based approach is shown (see fig. 3, 4 and 5) that incorporates the partial version selection of fig. 7, where the total valency is four, but the specificity is limited to two specificities. The N-terminus is composed of two identical soluble VH domains based on the VH3 germline, and the c-terminus is composed of two identical soluble VH domains based on the VH3 germline, the latter domains having different specificities than the N-terminal domain. Thus, each specificity has a valence of two. In addition, in this version, any antibody moiety at any of the four points of attachment/fusion may be replaced by a non-antibody moiety, e.g., not comprising an effector binding/modulating portion of an antibody molecule.

Fig. 10 illustrates another example. In this example, the molecule consists of four soluble VH domains based on the VH3 germline. The first two domains have the same specificity (e.g., inhibitory receptors), the 3 rd domain from the N-terminus may be specific for tissue antigens, and the fourth domain from the N-terminus may be specific for Human Serum Albumin (HSA), conferring an extended half-life to the molecule in the absence of the Ig Fc domain. There are three glycine, serine, alanine and/or threonine rich linkers between domains 1 and 2, domains 2 and 3 and domains 3 and 4. This version can be configured with up to four specificities in each case, but monovalent, or with bi-specificities in each case, bivalent. The order of the domains may be changed. In addition, in this version, any antibody moiety may be substituted with a non-antibody moiety, e.g., not comprising an effector binding/modulating portion of an antibody molecule.

Fig. 11 illustrates yet another method. This example is similar to fig. 3 and 4, as it is based on an Fc homodimer with two identical Fab units at the N-terminus of the molecule (bivalent monospecificity). This example differs in that each heavy chain is attached at its C-terminus with a tandem scFv. Thus, in each case, the C-terminus of the CH3 domain of the Fc is linked by a glycine/serine/alanine/threonine based linker to the N-terminus of a first VH domain linked via the C-terminus by a 12-15 amino acid glycine/serine rich linker to the N-terminus of a first VL domain linked C-terminally by a 25-35 amino acid glycine/serine/alanine/threonine linker to the N-terminus of a second VH domain linked via the C-terminus with a 12-15 amino acid glycine/serine based linker to the N-terminus of a second VL domain. In this Fc homodimer-based molecule, there are thus two identical tandem scfvs at the c-terminus of the molecule that provide any single valency (e.g., tetravalent) for a single tissue antigen, or two valencies for two different molecules. This type of format can also be adapted to a heterodimeric Fc core, which allows two different tandem scfvs at the c-terminus of the Fc to allow monovalent tetraspecificity at the c-terminus while retaining bivalent monospecificity at the N-terminus or monovalent bispecific at the N-terminus by using a single chain Fab configuration as in fig. 5, 6 and 7. The molecule may thus be configured to have 2, 3, 4, 5 or 6 specificities. The domain order of the scFv can be configured within the tandem-scFv unit from N to C-terminus as VH-linker-VL or VL-linker-VH. In addition, in this version, any antibody moiety at any of the four points of attachment/fusion may be replaced by a non-antibody moiety, e.g., not comprising an effector binding/modulating portion of an antibody molecule.

Bispecific antibodies can also be constructed with, for example, shorter systemic PK, with increased tissue penetration. These types of antibodies can be based, for example, on a domain antibody pattern based on human VH 3. These are illustrated in, for example, fig. 12, 13 and 14. Figures 12, 13 and 14 each comprise soluble VH domain modules based on the VH3 germline family. Each domain is approximately 12.5kDa, allowing for a small total MW, which, without being bound by any particular theory, should be beneficial for enhanced tissue penetration. In these examples, none of the VH domains recognize any half-life extended target, such as FcRn or HSA. As shown in figure 12, this molecule consists of two VH domains linked together by a flexible hydrophilic glycine/serine based linker between the C-terminus of the first domain and the N-terminus of the second domain. In this example, one domain may recognize a T cell costimulatory receptor and the second domain may recognize a tissue tethering antigen. As shown in fig. 13, the molecule consists of three VH domains, and an N-C terminal connection of a glycine/serine based hydrophilic linker. The molecules can be configured to be trispecific but monovalent for each target. It may be bispecific, bivalent to one target and monovalent to the other target. As shown in figure 14, the molecule consists of four VH domains with a glycine/serine rich N-C terminal linker between each domain. The molecules can be configured as tetraspecific, trispecific or bispecific, in each case with different antigenic valences. In addition, in this version, any antibody moiety may be substituted with a non-antibody moiety, e.g., not comprising an effector binding/modulating portion of an antibody molecule.

Other embodiments of bispecific antibodies are shown in figures 15 and 16. FIGS. 15 and 16 are composed of a natural heterodimeric core of the human IgG CH1/Ckappa interface, including a c-terminal heavy/light disulfide bond that covalently anchors the interaction. This format does not contain Fc or any moiety for half-life extension. As shown in FIG. 15, the molecule is appended at the N-terminus of the constant kappa domain with an scFv fragment consisting of an N-terminal VH domain linked at its C-terminus to the N-terminus of the VL domain via a gly/ser linker based on 12-15 amino acids, linked at 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 to the N-terminus of a VH domain linked at its c-terminus to the N-terminus of the CH1 domain by a native VH-CH1 elbow sequence. As shown in fig. 16, this molecule has the same N-terminal configuration as example 13. However, the C-terminus of the constant kappa and CH1 domains is appended with an scFv module, which may be in a VH-VL or VL-VH configuration, and may be specific for the same antigen or for two different antigens. The VH/VL interdomain linker may be 12-15 amino acids in length and consist of gly/ser residues. The scFv binding subunit can be exchanged for a soluble VH domain, or a peptide recognition element, or even a tandem-scFv element. The method may also be configured to use constant lambda and/or variable lambda domains. In addition, in this version, any antibody moiety at any one of the points of attachment/fusion may be replaced by a non-antibody moiety, e.g., not comprising an effector binding/modulating portion of 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 using a 12-15 amino acid gly/ser rich linker, which is then linked at the first VH C-terminus to the N-terminus of a second VL domain via a 25-30 amino acid gly/ser/ala/thr based linker. The second VL domain is linked at the C-terminus to the N-terminus of the second VH domain by a 12-15 amino acid gly/ser linker. Each scFv recognizes a different target antigen, such as costimulatory T cell molecules and tissue tethering targets. In addition, in this version, any antibody moiety may be substituted with a non-antibody moiety, e.g., not comprising an effector binding/modulating portion of an antibody molecule.

Fig. 18 illustrates another embodiment. FIG. 18 is a F (ab') 2scFv fusion. It consists of two identical Fab components linked by two disulfide bonds in the c-terminus of the native human IgG1 hinge region of the human IgG CH1 domain. Human IgG1CH2 and CH3 domains were absent. At the c-terminus of heavy chains 1 and 2 are two identical scFv fragments linked to the c-terminus of the huIgG1 hinge region by a gly/ser/ala/thr rich linker. In the configuration shown, the VH is N-terminal in each scFv unit and is linked to the N-terminus of the VL domain by a linker rich in 12-15 amino acids gly/ser. An alternative configuration is the N-terminal VL-linker-VH-C terminal. In this design, the construct is bispecific, being bivalent to the access target. In addition, in this version, any antibody moiety at any of the four points of attachment/fusion may be replaced by a non-antibody moiety, e.g., not comprising an effector binding/modulating portion of an antibody molecule.

The term CD39 molecule as used herein refers to a polypeptide having sufficient CD39 sequence to phosphohydrolyze ATP to AMP as part of a therapeutic compound. In some embodiments, the CD39 molecule phosphorylates ATP to AMP, and the CD39 molecule is identical or at least identical to 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95% of naturally occurring CD39 (e.g., CD39 from which the CD39 molecule was derived). In some embodiments, the CD39 molecule has at least 60, 70, 80, 90, 95, 99, or 100% sequence identity or substantial sequence identity to naturally occurring CD 39.

Any functional isoform (for CD39 or other proteins discussed herein) may be used. Exemplary CD39 sequences include the genbank accession number NP _001767.3 or mature forms from the following sequences:

MEDTKESNVKTFCSKNILAILGFSSIIAVIALLAVGLTQNKALPENVKYGIVLDAGSSHTSLYIYKWPAEKENDTGVVHQVEECRVKGPGISKFVQKVNEIGIYLTDCMERAREVIPRSQHQETPVYLGATAGMRLLRMESEELADRVLDVVERSLSNYPFDFQGARIITGQEEGAYGWITINYLLGKFSQKTRWFSIVPYETNNQETFGALDLGGASTQVTFVPQNQTIESPDNALQFRLYGKDYNVYTHSFLCYGKDQALWQKLAKDIQVASNEILRDPCFHPGYKKVVNVSDLYKTPCTKRFEMTLPFQQFEIQGIGNYQQCHQSILELFNTSYCPYSQCAFNGIFLPPLQGDFGAFSAFYFVMKFLNLTSEKVSQEKVTEMMKKFCAQPWEEIKTSYAGVKEKYLSEYCFSGTYILSLLLQGYHFTADSWEHIHFIGKIQGSDAGWTLGYMLNLTNMIPAEQPLSTPLSHSTYVFLMVLFSLVLFTVAIIGLLIFHKPSYFWKDMV(SEQ ID NO:1)。

in some embodiments, the 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 bloodestream mediated activities of soluble adenylate kinase-1 and NTPDase1/CD39 activities, yegotkin et al fasseb j.2012, month 9; 26(9):3875-83. Soluble recombinant CD39 fragments are also described in Inhibition of platelet function by recombinant soluble ecto-ADPase/CD39, Gayle et al, J Clin invest.1998, 5.1.d; 101(9):1851-1859.

The term CD73 molecule as used herein refers to a polypeptide having a sufficient CD73 sequence to dephosphorylate extracellular AMPs to adenosine as part of a therapeutic compound. In some embodiments, the CD73 molecule dephosphorylates extracellular AMP to adenosine, and the CD73 molecule is identical or at least identical to 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95% of naturally occurring CD73 (e.g., CD73 from which the CD73 molecule was derived). In some embodiments, the CD73 molecule has at least 60, 70, 80, 90, 95, 99, or 100% sequence identity or substantial sequence identity to naturally occurring CD 73. Exemplary CD73 sequences include the genes bank AAH 65937.15' -nucleotidase, ecto (CD73) [ homo sapiens ] or mature forms from the following sequences:

MCPRAARAPATLLLALGAVLWPAAGAWELTILHTNDVHSRLEQTSEDSSKCVNASRCMGGVARLFTKVQQIRRAEPNVLLLDAGDQYQGTIWFTVYKGAEVAHFMNALRYDAMALGNHEFDNGVEGLIEPLLKEAKFPILSANIKAKGPLASQISGLYLPYKVLPVGDEVVGIVGYTSKETPFLSNPGTNLVFEDEITALQPEVDKLKTLNVNKIIALGHSGFEMDKLIAQKVRGVDVVVGGHSNTFLYTGNPPSKEVPAGKYPFIVTSDDGRKVPVVQAYAFGKYLGYLKIEFDERGNVISSHGNPILLNSSIPEDPSIKADINKWRIKLDNYSTQELGKTIVYLDGSSQSCRFRECNMGNLICDAMINNNLRHADETFWNHVSMCILNGGGIRSPIDERNNGTITWENLAAVLPFGGTFDLVQLKGSTLKKAFEHSVHRYGQSTGEFLQVGGIHVVYDLSRKPGDRVVKLDVLCTKCRVPSYDPLKMDEVYKVILPNFLANGGDGFQMIKDELLRHDSGDQDINVVSTYISKMKVIYPAVEGRIKFSTGSHCHGSFSLIFLSLWAVIFVLYQ(SEQ ID NO:2)。

in some embodiments, the CD73 molecule comprises a soluble form of CD73, which can be shed from endothelial cell membranes by proteolytic cleavage or hydrolysis of the GPI anchor using shear stress, see, e.g., references: yegutkin G, Bodin P, Burnstock G.Effect of shear stress on the release of soluble ecto-enzymesatPase and 5' -nucleotidase binding with endogenous ATP from vascular and other cells Br J Pharmacol 2000; 129:921-6. For CD73 function, see Colgan et al, Physiological roles for ecto-5' -nucleotidase (CD73), Purinergic Signalling, 6.2006, 2: 351.

As used herein, a cell surface molecule binding agent refers to a molecule, typically a polypeptide, which (e.g. specifically) binds to a cell surface molecule on a cell (e.g. an immunosuppressive immune cell, such as a Treg). In some embodiments, the cell surface binding agent has sufficient sequence of a naturally occurring ligand from a cell surface molecule that can specifically bind to a cell surface molecule (cell surface molecule ligand). In some embodiments, the cell surface binding is antibody molecule binding, e.g., specific binding to a cell surface molecule.

As used herein, the term donor-specific targeting moiety refers to a moiety, such as an antibody molecule, that is a component of a therapeutic compound that preferentially locates the therapeutic compound to implanted donor tissue relative to the recipient's tissue. As a component of a therapeutic compound, a donor-specific targeting moiety provides a site-specific immune-privileged for a transplanted tissue (e.g., organ) from a donor.

In some embodiments, the donor-specific targeting moiety binds to a product (e.g., a polypeptide product) of an allele present at a locus that is not present in the locus of the (recipient) subject. In some embodiments, the donor-specific targeting moiety binds to an epitope on the product that is not present in the (recipient) subject.

In some embodiments, the donor-specific targeting moiety, as a component of the therapeutic compound, preferentially binds to the donor target or antigen, e.g., has a binding affinity for the donor target that is greater than, e.g., at least 2, 4, 5, 10, 50, 100, 500, 1,000, 5,000, or 10,000 times greater than, the binding affinity of the donor-specific targeting moiety to the subject antigen or tissue. In some embodiments, the binding affinity of the donor-specific targeting moiety to the product of an allele of a locus present in the donor tissue (but not present in the subject) is at least 2, 4, 5, 10, 50, 100, 500, 1,000, 5,000, or 10,000 times greater than its affinity to the product of an allele of a locus present in the subject (which allele is not present in the donor tissue). The affinity of a therapeutic compound for which the donor-specific moiety is a component can be measured in a cell suspension, for example, by comparing the affinity for suspension cells having an allele with the affinity of a therapeutic compound for suspension cells not having an allele. In some embodiments, the binding affinity to the donor allele cell is less than 10 nM. In some embodiments, the binding affinity to the donor allele cell is less than 100pM, 50pM, or 10 pM.

In some embodiments, the specificity for the donor allele product is sufficient for the donor-specific targeting moiety, when coupled with an immune down-regulating effector: i) immune attack of the implanted tissue (e.g., as measured by histological inflammatory responses), infiltrating T effector cells, or organ function in a clinical setting-e.g., creatinine to the kidney is significantly reduced, e.g., compared to that seen in an otherwise similar implant lacking a donor-specific targeting moiety coupled with an immune down-regulating effector; and/or ii) substantially maintaining immune function in the recipient external to or remote from the implanted tissue. In some embodiments, one or more of the following can be seen: peripheral blood lymphocyte counts are not substantially affected at therapeutic levels of the therapeutic compound, e.g., T cell levels are within 25%, 50%, 75%, 85%, 90%, or 95% of normal, B cell levels are within 25%, 50%, 75%, 85%, 90%, or 95% of normal, and/or granulocyte (PMN) cell levels are within 25%, 50%, 75%, 85%, 90%, or 95% of normal, or monocyte levels are within 25%, 50%, 75%, 85%, 90%, or 95% of normal; the in vitro proliferative function of PBMCs (peripheral blood mononuclear cells) for non-disease associated antigens is essentially normal or within 70%, 80% or 90% of normal at therapeutic levels of the therapeutic compound; the incidence or risk of opportunistic infections and cancer associated with immunosuppression is not substantially increased compared to normal at therapeutic levels of the therapeutic compound; or at therapeutic levels of therapeutic compounds, the incidence or risk of opportunistic infections and cancer associated with immunosuppression is significantly lower than that seen with standard-of-care or non-targeted immunosuppression. In some embodiments, the donor-specific targeting moiety comprises an antibody molecule, a target-specific binding polypeptide, or a target ligand binding molecule.

As used herein, an effector refers to an entity that mediates an immune response, such as a cell or molecule, e.g., a soluble or cell surface molecule.

As used herein, an effector ligand binding molecule refers to a polypeptide having sufficient sequence of naturally occurring anti-ligands from an effector that can bind specifically enough to make it available as an effector binding/modulating molecule. In some embodiments, it binds to the effector with at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the affinity of the naturally-occurring anti-ligand. In some embodiments, it has at least 60, 70, 80, 90, 95, 99, or 100% sequence identity or substantial sequence identity to the naturally occurring counter-ligand of the effector.

As used herein, an effector specific binding polypeptide refers to a polypeptide that is capable of binding with sufficient specificity such that it can function as an effector binding/modulating moiety. In some embodiments, the specific binding polypeptide comprises an effector ligand binding molecule.

As used herein, elevated risk refers to the risk of a disorder in a subject, wherein the subject has one or more of: a history of a disorder or symptom of a disorder, a biomarker associated with a disorder or symptom of a disorder, or a family history of a disorder or symptom of a disorder.

As used herein, a functional antibody molecule to an effector or inhibitory immune checkpoint molecule refers to an antibody molecule that can bind to and agonize an effector or inhibitory immune checkpoint molecule when present as an ICIM binding/modulating portion of a multimerizing therapeutic compound. In some embodiments, the anti-effector or inhibitory immune checkpoint molecule antibody molecule, when bound as a monomer (or when the therapeutic compound is not multimerized) to an effector or inhibitory immune checkpoint molecule, does not antagonize, does not substantially antagonize, prevents or prevents substantial binding of an endogenous ligand of the inhibitory immune checkpoint molecule to the inhibitory immune checkpoint molecule. In some embodiments, the anti-effector or inhibitory immune checkpoint molecule antibody molecule, when bound as a monomer (or when the therapeutic compound is not multimerized) to the anti-inhibitory immune checkpoint molecule, does not agonize or substantially agonize the effector or inhibitory molecule.

As used herein, the term ICIM binding/modulating moiety refers to an effector binding/modulating moiety that, as part of a therapeutic compound, binds to and agonizes a cell surface inhibitory molecule, such as an inhibitory immune checkpoint molecule (e.g., PD-1), or binds to or modulates cell signaling, such as binds to FCRL (e.g., FCRL1-6), or binds to and antagonizes a molecule that promotes immune function.

As used herein, the term IIC binding/modulating moiety refers to an effector binding/modulating moiety that binds to an immunosuppressive immune cell as part of a therapeutic compound. In some embodiments, the IIC binding/modulating moiety increases the number or concentration of immunosuppressive immune cells at the binding site.

The term "inhibitory immune checkpoint molecule ligand molecule" as used herein refers to a polypeptide having sufficient inhibitory immune checkpoint molecule ligand sequence (e.g., sufficient PD-L1 sequence in the case of PD-L1 molecules) to bind to and agonize its cognate inhibitory immune checkpoint molecule (e.g., again PD-1 in the case of PD-L1) when present as an ICIM binding/modulating moiety of a multimeric therapeutic compound.

In some embodiments, the inhibitory immune checkpoint molecule ligand molecule (e.g., PD-L1 molecule), when bound as a monomer (or bound when the therapeutic compound is not multimerized) to its cognate ligand (e.g., PD-1), does not antagonize or substantially antagonizes the binding of the endogenous inhibitory immune checkpoint molecule ligand to the inhibitory immune checkpoint molecule, or prevents the binding of the endogenous inhibitory immune checkpoint molecule ligand to the inhibitory immune checkpoint molecule or prevents substantial binding. For example, with respect to the PD-L1 molecule, the PD-L1 molecule does not antagonize the binding of endogenous PD-L1 to PD-1.

In some embodiments, the inhibitory immune checkpoint molecule ligand, when bound as a monomer to its cognate inhibitory immune checkpoint molecule, does not agonize or does not substantially agonize the inhibitory immune checkpoint molecule. For example, a PD-L1 molecule, when bound to PD-1, does not agonize or does not substantially agonize PD-1, for example.

In some embodiments, the inhibitory immune checkpoint molecule ligand molecule has at least 60, 70, 80, 90, 95, 99 or 100% sequence identity or substantial sequence identity to the naturally occurring inhibitory immune checkpoint molecule ligand.

Exemplary inhibitory immune checkpoint molecule ligand molecules include: a PD-L1 molecule that binds to an inhibitory immune checkpoint molecule PD-1, and in embodiments has at least 60%, 70%, 80%, 90%, 95%, 99% or 100% sequence identity or substantial sequence identity to a naturally occurring PD-L1, such as a PD-L1 molecule comprising the sequence:

MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET (SEQ ID NO:3), or an active fragment thereof; in some embodiments, the active fragment comprises residues 19 to 290 of the PD-L1 sequence; an HLA-G molecule that binds to any one of the inhibitory immune checkpoint molecules KIR2DL4, LILRB1 and LILRB2, and in embodiments has at least 60, 70, 80, 90, 95, 99 or 100% sequence identity to a naturally occurring HLA-G. Exemplary HLA-G sequences include, for example, those found in genbank P17693.1 RecName: Full-HLA class I histocompatibility antigen, α chain G; the AltName: Full-HLA G antigen; the AltName: Full-MHC class I antigen G; flag: the sequence in the precursor, or the mature form found in sequence MVVMAPRTLFLLLSGALTLTETWAGSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRMNLQTLRGYYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRADPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWKQSSLPTIPIMGIVA (SEQ ID NO: 4).

As used herein, an inhibitory molecule anti-ligand molecule refers to a polypeptide having sufficient inhibitory molecule anti-ligand sequence such that it can bind to and activate a cognate inhibitory molecule when present as an ICIM binding/modulating moiety of a multimerization therapeutic compound. In some embodiments, the inhibitory molecule anti-ligand molecule, when bound to the inhibitory molecule as a monomer (or when the therapeutic compound is not multimerized), does not antagonize, does not substantially antagonize, prevents or substantially prevents substantial binding of the endogenous anti-ligand of the inhibitory molecule to the inhibitory molecule. In some embodiments, the inhibitory molecule anti-ligand molecule does not antagonize or substantially antagonize the inhibitory molecule when bound to the inhibitory molecule as a monomer (or when the therapeutic compound is not multimerized).

As used herein, sequence identity, percent identity, and related terms refer to the relatedness of two sequences, e.g., two nucleic acid sequences or two amino acid or polypeptide sequences. In the context of amino acid sequences, the term "substantially identical" is used herein to refer to a first amino acid containing a sufficient or minimum number of amino acid residues that are i) identical to an aligned amino acid residue in a second amino acid sequence or ii) conservative substitutions of an aligned amino acid residue in the second amino acid sequence such that the first and second amino acid sequences may have a common domain and/or common functional activity. For example, an amino acid sequence contains a common domain that has 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).

The term "substantially identical" is used herein in the context of a nucleotide sequence to refer to a first nucleic acid sequence containing 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 polypeptides having a common functional activity, or encode a common structural polypeptide domain or common functional polypeptide activity. For example, a nucleotide sequence has 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).

The term "functional variant" refers to a polypeptide that has substantially the same amino acid sequence as, or is encoded by substantially the same nucleotide sequence as, a naturally occurring sequence, and is capable of having one or more activities of the naturally occurring sequence.

The calculation of homology or sequence identity between sequences (these terms are used interchangeably herein) is performed as follows.

To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps are introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences can be omitted for comparison purposes). In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein, amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology").

The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap.

Comparison of sequences and determination of percent identity between two sequences can be accomplished using mathematical algorithms. In a preferred embodiment, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch ((1970) J.mol.biol.48:444-453) algorithm in the GAP program already incorporated into the GCG software package (available at www.gcg.com), using either the Blossom 62 matrix or the PAM250 matrix, together with the GAP weights of 16, 14, 12, 10, 8,6, or 4 and the length weights of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at www.gcg.com), using the nwsgapdna. cmp matrix and GAP weights of 40, 50, 60, 70, or 80 and length weights of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and parameters that should be used unless otherwise specified) is a Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

The percent identity between two amino acid nucleotide sequences can also be determined using the algorithms of e.meyers and w.miller ((1989) cabaos, 4:11-17) which have 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.

The nucleic acid and protein sequences described herein can be used as "query sequences" to search public databases, for example, to identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs of Altschul et al (1990) J.mol.biol.215:403-10 (version 2.0). A BLAST nucleotide search can be performed with the NBLAST program with a score of 100 and a word length of 12 to obtain nucleotide sequences that are homologous to any of the nucleic acid sequences provided herein, for example. BLAST protein searches can be performed using the XBLAST program with a score of 50 and a word length of 3 to obtain amino acid sequences that are homologous to the protein molecules provided herein, for example. To obtain gap alignments for comparison purposes, Gapped BLAST as described in Altschul et al, (1997) Nucleic Acids Res.25: 3389-. When BLAST and Gapped BLAST programs are used, the default parameters of the corresponding programs (e.g., XBLAST and NBLAST) can be used. See http:// www.ncbi.nlm.nih.gov.

As used herein, the term "hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions" describes conditions for hybridization and washing. Guidance for performing hybridization reactions can be found in Current protocols Molecular Biology, John Wiley & Sons, N.Y. (1989),6.3.1-6.3.6, which is incorporated herein by reference. Aqueous and non-aqueous methods are described in this reference, and either may be used. The specific hybridization conditions mentioned herein are as follows: 1) low stringency hybridization conditions are washing twice in 6X sodium chloride/sodium citrate (SSC) at about 45 ℃ followed by at least 50 ℃ in 0.2X SSC, 0.1% SDS (for low stringency conditions, the temperature of the wash may be raised to 55 ℃); 2) moderate stringency hybridization conditions are one or more washes in 6 XSSC at about 45 ℃ followed by 0.2 XSSC, 0.1% SDS at 60 ℃; 3) high stringency hybridization conditions are one or more washes in 6 XSSC at about 45 ℃ followed by 0.2 XSSC, 0.1% SDS at 65 ℃; and preferably 4) very high stringency hybridization conditions are 0.5M sodium phosphate, 7% SDS at 65 ℃, followed by one or more washes in 0.2 XSSC, 1% SDS at 65 ℃. Unless otherwise stated, the very high stringency conditions (4) are the preferred conditions and one should use.

It is understood that molecules and compounds of embodiments of the invention may have additional conservative or non-essential amino acid substitutions that do not significantly affect their function.

The term "amino acid" is intended to include all molecules, whether natural or synthetic, which include amino functionality and acid functionality, and can be included in polymers of naturally occurring amino acids. Exemplary amino acids include naturally occurring amino acids; analogs, derivatives and homologs thereof; amino acid analogs having variant side chains; and all stereoisomers of any of the foregoing. As used herein, the term "amino acid" includes 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 with similar side chains have been defined in the art. These families include amino acids with the following side: 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). A CD39 molecule, a CD73 molecule, a cell surface molecule binding agent, a donor-specific targeting moiety, an effector ligand binding molecule, an ICIM binding/modulating moiety, an IIC binding/modulating moiety, an inhibitory immune checkpoint molecule ligand molecule, an inhibitory molecule anti-ligand molecule, an SM binding/modulating moiety.

As used herein, the term SM binding/modulating moiety refers to the following effector binding/modulating moieties: promoting an immunosuppressive local microenvironment as part of a therapeutic compound, for example by providing a substance proximal to the target that inhibits or minimizes the attack of the targeted immune system. In some embodiments, the SM binding/modulating moiety comprises or binds to a molecule that inhibits or minimizes immune system attack of the target. In some embodiments, the therapeutic compound comprises an SM binding/modulating moiety that binds to and accumulates soluble substances with immunosuppressive functions, such as endogenous or exogenous substances. In some embodiments, the therapeutic compound comprises an SM binding/modulating moiety that binds to and inhibits, sequesters, degrades or otherwise neutralizes a substance that promotes immune attack, typically an endogenous soluble molecule. In some embodiments, the therapeutic compound comprises an SM binding/modulating moiety comprising an immunosuppressive substance, such as a protein fragment known to be immunosuppressive. For example, the effector molecule binding moiety binds to or comprises a substance (e.g., a CD39 molecule or a CD73 molecule) that depletes a component (e.g., ATP or AMP) that promotes immune effector cell function.

As used herein, a specific targeting moiety refers to a donor-specific targeting moiety or a tissue-specific targeting moiety.

As used herein, a subject refers to a mammalian subject, e.g., a human subject. In some embodiments, the subject is a non-human mammal, such as a horse, dog, cat, cow, goat, or pig.

As used herein, a target ligand binding molecule refers to a polypeptide having sufficient sequence of a naturally occurring anti-ligand from a target ligand that can bind with sufficient specificity to the target ligand such that it can act as a specific targeting moiety. In some embodiments, it binds to the target tissue or cell with at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the affinity of the naturally-occurring anti-ligand. In some embodiments, it has at least 60, 70, 80, 90, 95, 99, or 100% sequence identity or substantial sequence identity to the naturally occurring counter-ligand of the target ligand.

As used herein, a target site refers to a site that contains an entity (e.g., an epitope) bound by a targeting moiety. In some embodiments, the target site is a site that establishes immune-privileged immunity.

As used herein, a tissue-specific targeting moiety refers to a moiety, such as an antibody molecule, that is a component of a therapeutic molecule that preferentially localizes the component of the therapeutic molecule to a target tissue relative to other tissues of a subject. As a component of the therapeutic compound, the tissue-specific targeting moiety provides site-specific immune-privileged tissue, e.g., a target tissue, such as an organ or tissue, that is subject to or at risk of immune attack. In some embodiments, the tissue-specific targeting moiety binds to a product, e.g., a polypeptide product, that is not present outside the target tissue, or is present at a sufficiently low level such that at a therapeutic concentration of the therapeutic molecule, an unacceptable level of immunosuppression is not present or is substantially absent. In some embodiments, the tissue-specific targeting moiety binds to an epitope that is not present or substantially not present outside the target site.

In some embodiments, the tissue-specific targeting moiety, as a component of the therapeutic compound, preferentially binds to the target tissue or target antigen, e.g., has a binding affinity for the target tissue or antigen that is greater than, e.g., at least 2, 4, 5, 10, 50, 100, 500, 1,000, 5,000, or 10,000 times greater than, the binding affinity of the tissue-specific targeting moiety for non-target tissues or antigens present outside of the target tissue. The affinity of a therapeutic compound having a tissue-specific moiety as a component thereof can be measured in a cell suspension, for example, by comparing the affinity of the therapeutic compound for suspension cells having a target antigen to the affinity of suspension cells not having the target antigen. In some embodiments, the binding affinity to cells carrying the target antigen is less than 10 nM.

In some embodiments, the binding affinity to cells carrying the target antigen is less than 100pM, 50pM, or 10 pM. In some embodiments, the specificity for the target antigen is sufficient for the tissue-specific targeting moiety to couple with an immune down-regulating effector: i) immune attack of the target tissue (e.g., as measured by a histological inflammatory response), infiltrating T effector cells, or organ function in a clinical setting-e.g., creatinine in the kidney is significantly reduced, e.g., compared to that seen in an otherwise similar implant lacking tissue-specific targeting moieties coupled with immune down-regulating effectors; and/or ii) substantially maintaining immune function in the recipient external to or remote from the target tissue.

In some embodiments, one or more of the following can be seen: peripheral blood lymphocyte counts are not substantially affected at therapeutic levels of the therapeutic compound, e.g., T cell levels are within 25%, 50%, 75%, 85%, 90%, or 95% of normal, B cell levels are within 25%, 50%, 75%, 85%, 90%, or 95% of normal, and/or granulocyte (PMN) cell levels are within 25%, 50%, 75%, 85%, 90%, or 95% of normal, or monocyte levels are within 25%, 50%, 75%, 85%, 90%, or 95% of normal; the in vitro proliferative function of PBMCs (peripheral blood mononuclear cells) for non-disease associated antigens is essentially normal or within 70%, 80% or 90% of normal at therapeutic levels of the therapeutic compound; the incidence or risk of opportunistic infections and cancer associated with immunosuppression is not substantially increased compared to normal at therapeutic levels of the therapeutic compound; or at therapeutic levels of therapeutic compounds, the incidence or risk of opportunistic infections and cancer associated with immunosuppression is significantly lower than that seen with standard therapy or non-targeted immunosuppression. In some embodiments, the tissue-specific targeting moiety comprises an antibody molecule. In some embodiments, the donor-specific targeting moiety comprises an antibody molecule, a target-specific binding polypeptide, or a target ligand binding molecule. In some embodiments, the tissue-specific targeting moiety binds to the product or to a site on the product that is present or expressed only on the target tissue or substantially only on the target tissue.

ICIM binding/modulating moiety: effector binding/modulating moieties that bind inhibitory receptors

The methods and compounds described herein provide therapeutic compounds having an effector binding/modulating moiety comprising an ICIM binding/modulating moiety that directly binds to and activates an inhibitory receptor on the surface of an immune cell, e.g., to reduce or eliminate, or substantially eliminate the ability of an immune cell to mediate immune attack. Coupling of the ICIM binding/modulating moiety to the targeting entity facilitates site-specific or local down-regulation of the immune cell response, e.g., substantially restricted to the location of the binding site with the targeting moiety. Thus, normal systemic immune function is substantially retained. In some embodiments, the ICIM binding/modulating moiety comprises an inhibitory immune checkpoint molecule anti-ligand molecule of an inhibitory immune checkpoint molecule, e.g., a natural ligand, or a fragment of a natural ligand (e.g., PD-L1 or HLA-G). In some embodiments, the ICIM binding/modulating moiety comprises a functional antibody molecule that engages an inhibitory immune checkpoint molecule, for example a functional antibody molecule comprising a scFv binding domain.

In some embodiments, an ICIM binding/modulating moiety comprising, for example, a functional antibody molecule or an inhibitory immune checkpoint molecule ligand molecule binds to an inhibitory receptor but does not prevent binding of the inhibitory receptor's natural ligand to the inhibitory receptor. In embodiments, the following patterns are used: wherein the targeting moiety is coupled (e.g., fused) to an ICIM binding/modulating moiety comprising, for example, an scFv domain, and is configured such that upon binding of the inhibitory receptor in solution (e.g., blood or lymph) (and possibly in monomeric form), the therapeutic molecule: i) (ii) does not agonize, or substantially does not agonize (e.g., agonize at 30%, 20%, 15%, 10%, or 5% below the levels seen with fully agonistic molecules) inhibitory receptors on immune cells; and/or ii) fails to antagonize or substantially fails to antagonize (e.g., antagonizes at 30%, 20%, 15%, 10%, or 5% below the levels seen with a fully antagonistic molecule) inhibitory receptors on immune cells. Candidate molecules can be assessed for their ability to agonize or not by their ability to increase or decrease an immune response in a cell-based in vitro assay in which the target is not expressed, e.g., using an MLR-based assay (mixed lymphocyte reaction).

In some embodiments, a candidate ICIM binding/modulating moiety may reduce, completely or substantially eliminate systemic immunosuppression and systemic immune activation. In some embodiments, the targeting domain of the therapeutic compound, when bound to the target, will serve to aggregate or multimerize the therapeutic compound on the surface of the tissue in need of immune protection. In some embodiments, an ICIM binding/modulating moiety (e.g., an ICIM binding/modulating moiety comprising an scFv domain) requires an aggregation or multimerization state to be able to deliver agonistic and immunosuppressive signals, or a multitude of such signals, to local immune cells. Therapeutic agents of this type may, for example, provide local immunosuppression while leaving the systemic immune system undisturbed or substantially undisturbed. That is, immunosuppression is localized to the location where it is desired, rather than being systemic and not localized to a specific area or tissue type.

In some embodiments, upon binding to a target (e.g., a target organ, tissue, or cell type), the therapeutic compound coats the target, e.g., the target organ, tissue, or cell type. The therapeutic agent provides a "masking" signal only at the site of therapeutic compound accumulation or to a greater extent at the site of therapeutic compound accumulation when circulating lymphocytes attempt to bind to and destroy the target.

The ability of a candidate therapeutic compound to bind (e.g., specifically bind) to its target can be assessed, for example, by ELISA, cell-based assays, or surface plasmon resonance. This property should generally be maximized because of its site-specific and local nature that mediates immune-privileged immunity. The ability of a candidate therapeutic compound to down-regulate immune cells upon binding to a target can be assessed, for example, by a cell-based activity assay. This property should generally be maximized because of its site-specific and local nature that mediates immune-privileged immunity. The level of down-regulation achieved by a candidate therapeutic compound in monomeric (or non-binding) form can be assessed, for example, by cell-based activity assays. This property should generally be minimized because it mediates a systemic down-regulation of the immune system. The level of antagonism of a cell surface inhibitory molecule (e.g., an inhibitory immune checkpoint molecule) achieved by a candidate therapeutic compound in monomeric (or non-binding) form can be assessed, for example, by cell-based activity assays. This property should generally be minimized because it mediates the undesired activation of the immune system. In general, the properties should be selected and balanced to produce a site-specific immune-privileged that is sufficiently stable without an unacceptable level of non-site-specific agonism or antagonism of inhibitory immune checkpoint molecules.

Exemplary inhibitory immune checkpoint molecules

Exemplary suppressor molecules (e.g., suppressor immune checkpoint molecules) (along with their anti-ligands) can be found in table 1. The table lists the molecules to which exemplary ICIM binding moieties can bind.

PD-L1/PD-1 pathway

Programmed cell death protein 1 (often referred to as PD-1) is a cell surface receptor belonging to the immunoglobulin superfamily. PD-1 is expressed on T cells and other cell types including, but not limited to, B cells, myeloid cells, dendritic cells, monocytes, T regulatory cells, and inkt cells. PD-1 binds two ligands, PD-L1 and PD-L2, and is an inhibitory immune checkpoint molecule. In the case where antigen-loaded MCH engages T cell receptors on T cells, engagement with the cognate ligand PD-L1 or PD-L2 minimizes or prevents T cell activation and function. Inhibition of PD-1 may include promotion of apoptosis (programmed cell death) of antigen-specific T cells in lymph nodes and reduction of apoptosis of regulatory T cells (suppressor T cells).

In some embodiments, the therapeutic compound comprises an ICIM binding/modulating moiety that agonizes PD-1 inhibition. The ICIM binding/modulating moiety may include an inhibitory molecular anti-ligand molecule, such as a ligand fragment comprising PD-1 (e.g., a fragment of PD-L1 or PD-L2); or another moiety, such as a functional antibody molecule, comprising, for example, a scFv domain that binds PD-1.

In some embodiments, the therapeutic compound comprises a targeting moiety that preferentially binds to a donor antigen that is not present in the subject, is present at significantly lower levels in the subject, e.g., a donor antigen of table 2, and is localized to donor transplant tissue in the subject. In some embodiments, it does not bind or does not substantially bind other tissues. In some embodiments, the therapeutic compound may include a targeting moiety that is specific for HLA-a2 and that specifically binds to donor allograft tissue but does not bind or does not substantially bind to host tissue. In some embodiments, a therapeutic compound comprises an ICIM binding/modulating moiety, such as an inhibitory molecular anti-ligand molecule, for example, comprising a ligand fragment of PD-1 (e.g., a fragment of PD-L1 or PD-L2); or another moiety, such as a functional antibody molecule, comprising, for example, a scFv domain that binds to PD-1, such that the therapeutic compound activates PD-1, for example, upon binding to a target. The therapeutic compound targets the allograft and provides a local immune boost to the allograft.

In some embodiments, a therapeutic compound comprises a targeting moiety that preferentially binds to an antigen of table 3 and localizes to a 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. In some embodiments, a therapeutic compound comprises an ICIM binding/modulating moiety, such as an inhibitory molecular anti-ligand molecule, for example, comprising a ligand fragment of PD-1 (e.g., a fragment of PD-L1 or PD-L2); or another moiety, such as a functional antibody molecule, comprising, for example, a scFv domain that binds to PD-1, such that the therapeutic compound activates PD-1, for example, upon binding to a target. The therapeutic compound targets a tissue subject to autoimmune attack and provides a local immune boost to the tissue.

PD-L1 and PDL2, or polypeptides derived therefrom, may provide candidate ICIM binding moieties. However, in monomeric form, for example, when a therapeutic compound is circulating in the blood or lymph, the molecule may have an undesirable effect of antagonizing the PD-L1/PD-1 pathway, and may only agonize the PD-1 pathway when clustered or multimerized on the surface of a target (e.g., a target organ). In some embodiments, a therapeutic compound comprises an ICIM binding/modulating moiety comprising a functional antibody molecule (e.g., scFv domain) that is inert or substantially inert to the PD-1 pathway in soluble form, but agonizes and drives inhibitory signals upon multimerization (via a targeting moiety) on the tissue surface.

HLA-G: KIR2DL4/LILRB1/LILRB2 pathway

KIR2DL4, LILRB1, and LILRB2 are inhibitory molecules found on T cells, NK cells, and bone marrow cells. HLA-G is the respective counterligand.

KIR2DL4 is also known AS CD158D, G9P, KIR-103AS, KIR103AS, KIR-2DL4, killer cell immunoglobulin-like receptor, and two Ig domains and long cytoplasmic tail 4. LILRB1 is also called LILRB1, CD85J, ILT-2, ILT2, LIR-1, LIR1, MIR-7, MIR7, PIR-B, PIRB, and leukocyte immunoglobulin-like receptor B1. LILRB2 is also known as CD85D, ILT-4, LIR-2, LIR2, MIR-10, MIR10, and ILT 4.

Therapeutic compounds comprising HLA-G molecules may be used to provide inhibitory signals to immune cells comprising any of KIR2DL4, LILRB1 and LILRB2, for example to provide multimerised therapeutic compound molecules comprising HLA-G molecules and thus to provide site-specific immune-privileged.

Therapeutic compounds comprising agonistic anti-KIR 2DL4, anti-LILRB 1, or anti-LILRB 2 antibody molecules are useful for providing inhibitory signals to immune cells comprising any of KIR2DL4, LILRB1, and LILRB 2.

HLA-G delivers inhibitory signals only upon multimerization, e.g., expression on the cell surface, or upon binding to the bead surface. In embodiments, therapeutic compounds are provided that comprise HLA-G molecules that do not multimerize in solution (or do not multimerize sufficiently to produce a significant level of inhibitory molecular agonism). The use of HLA-G molecules that minimize multimerization in solution will minimize systemic agonism and unwanted immunosuppression of immune cells.

While not wishing to be bound by theory, it is believed that: HLA-G does not function in down regulation unless multimerized; binding of the therapeutic compound to the target via the targeting moiety multimerizes the ICIM-binding entity; and the multimerized ICIM binding entity binds to and clusters inhibitory molecules at the surface of the immune cells, thereby mediating down-regulation of negative signaling by the immune cells. Thus, infiltrating immune cells, including antigen presenting cells and other bone marrow cells, NK cells, and T cells, that attempt to destroy the target tissue are down-regulated.

While it is desirable for HLA-G molecules to minimize antagonism when in monomeric form, the redundancy of LILRB1 and LILRB2 will minimize the effect on systemic antagonism (with some monomeric antagonism at all times).

In some embodiments, a therapeutic compound comprises an ICIM binding/modulating moiety comprising an HLA-G molecule, e.g., an isoform without B2M (e.g., HLA-G5), see Carosella et al, advanced sin Immunology,2015,127: 33. In the B2M-free form, HLA-G preferentially binds to LILRB 2.

Suitable sequences for constructing HLA-G molecules include genbank P17693.1 RecName: Full-HLA class I histocompatibility antigen, α chain G; the AltName: Full-HLA G antigen; the AltName: Full-MHC class I antigen G; flag: precursor, or MVVMAPRTLFLLLSGALTLTETWAGSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRMNLQTLRGYYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRADPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWKQSSLPTIPIMGIVAGLVVLAAVVTGAAVAAVLWRKKSSD (SEQ ID NO: 5). The suitability of candidate HLA-G molecules for use in methods and compounds may be tested, for example, by methods similar to those described in "Synthetic HLA-G proteins for therapeutic use in transplantation," LeMaoult et al, 2013The FASEB Journal 27:3643,

in some embodiments, the therapeutic compound comprises a targeting moiety that preferentially binds to a donor antigen that is not present in the subject, is present at significantly lower levels in the subject, e.g., a donor antigen of table 2, and is localized to donor transplant tissue in the subject. In some embodiments, it does not bind or does not substantially bind other tissues. In some embodiments, a therapeutic compound may include a targeting moiety that is specific for HLA-a2 and that specifically binds to donor allografts but not to host tissue, and that is combined with an ICIM binding/modulating moiety comprising an HLA-G molecule that binds KIR2DL4, LILRB1, or LILRB2, such that the therapeutic compound activates KIR2DL4, LILRB1, or LILRB2, e.g., upon binding to the target. The therapeutic compound targets the allograft and provides a local immune boost to the allograft.

In some embodiments, the therapeutic compound comprises a targeting moiety that preferentially binds to a tissue-specific antigen (e.g., an antigen of table 3) and is localized to a target site 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. In embodiments, a therapeutic compound comprises an ICIM binding/modulating moiety comprising an HLA-G molecule that binds KIR2DL4, LILRB1, or LILRB2, such that the therapeutic compound activates KIR2DL4, LILRB1, or LILRB2, e.g., upon binding to a target. The therapeutic compound targets a tissue subject to autoimmune attack and provides a local immune boost to the tissue.

It is possible to engineer stable soluble HLA-G-B2M fusion proteins which also bind to LILRB 1. For example, HLA-G/B2M monomer was used to determine the crystal structure of HLA-G (Clements et al 2005PNAS 102: 3360).

FCRL family

FCRL1-6 generally inhibits B cell activation or function. These type 1 transmembrane glycoproteins are composed of different combinations of 5 types of immunoglobulin-like domains, each protein consisting of 3 to 9 domains, and no single domain type is conserved among all FCRL proteins. In general, FCRL expression is limited to lymphocytes, and is primarily expressed in B lymphocytes. In general, FCRL is used to inhibit B cell activation.

The ICIM binding/modulating moiety may comprise an agonistic anti-BCMA antibody molecule. In some embodiments, the therapeutic compound comprises an anti-FCRL antibody molecule and an anti-B Cell Receptor (BCR) antibody molecule. While not wishing to be bound by theory, it is believed that a therapeutic compound comprising an antibody molecule with two specificities will bring the FCRL into close proximity with the BCR and inhibit BCR signaling.

Milk-like proteins and milk-like proteins

The effector binding/modulating moiety may comprise an agonist or antagonist of a lactotroph protein. In some embodiments, the effector binding/modulating moiety comprises an agonistic or functional BTN1a1 molecule, BTN2a2 molecule, BTNL2 molecule, or BTNL1 molecule.

As used herein, a functional BTNXi molecule (wherein Xi ═ 1a1, 2a2, L2, or L1) refers to a polypeptide having sufficient sequence of BTNXi to inhibit T cells as part of a therapeutic compound. In some embodiments, the BTNXi molecule has at least 60, 70, 80, 90, 95, 99, or 100% sequence identity or substantial sequence identity to a naturally occurring lactophin or lactophin-like molecule.

In some embodiments, the effector binding/modulating moiety comprises an antagonistic BTNL8 molecule.

As used herein, the term antagonistic BTNL8 molecule refers to a polypeptide having sufficient BTNL8 sequence to inhibit the activation, proliferation, or secretion of cytokines by resting T cells as part of a therapeutic compound. In some embodiments, the BTNL8 molecule has at least 60, 70, 80, 90, 95, 99, or 100% sequence identity or substantial sequence identity to a naturally occurring cremophilic protein.

IIC binding/modulating moiety: effector binding/modulating moieties for recruitment of immunosuppressive T cells

In some embodiments, the therapeutic compound comprises an effector binding/modulating moiety, e.g., an IIC binding/modulating moiety, that binds, activates or retains an immunosuppressive cell, e.g., an immunosuppressive T cell, at a site mediated by the targeting moiety, thereby providing site-specific immune-privileged. An IIC binding/modulating moiety, e.g., an IIC binding/modulating moiety comprising an antibody molecule (comprising, e.g., an scFv binding domain), binds an immunosuppressive cell type, e.g., a Treg, e.g., Foxp3+ CD25+ Treg. Organ, tissue or specific cell type tolerance is associated with a massive increase in proximal tregs and infiltration of the target organ; in embodiments, the methods and compounds described herein synthetically reconstruct and mimic this physiological state. After accumulation of tregs, an immunosuppressive microenvironment is established to protect the organ of interest from the immune system.

As T_REGAnd a GARP binding agent of a TGFB-targeting molecule

GARP is a membrane protein receptor for latent TGF- β expressed on the surface of activated tregs (Tran et al 2009PNAS 106:13445 and Wang et al 2009PNAS 106: 13439). In some embodiments, the therapeutic compound comprises an IIC binding entity that binds to 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 a target tissue of interest. IIC binding/modulating moieties comprising GARP-binding agents include, for example, IIC binding/modulating moieties comprising anti-GARP antibody molecules (e.g., anti-GARP scFv domains). While not wishing to be bound by theory, it is believed that therapeutic compounds comprising GARP binding agents effect accumulation of GARP-expressing tregs at the site targeted by the targeting portion of the therapeutic compound (e.g., the site of graft or organ injury). In addition, while not wishing to be bound by theory, it is believed that therapeutic compounds comprising the role of GARP binding agents may also achieve accumulation of soluble GARP at the site of organ injury that will serve to bind and activate the immunosuppressive cytokine TGFB1 in a localized manner (Fridrich et al 2016PLoS One 11: e 0153290; doi:10.1371/journal. po. 0153290 and Hahn et al 2013Blood 15: 1182). Thus, an effector binding/modulating moiety comprising a GARP binding agent may serve as an IIC binding/modulating moiety or an SM binding/modulating moiety.

As T_REGCTLA4 targeting and T-effector cell silencing molecules

In some embodiments, the effector binding/modulating moiety comprises, for example, an antibody molecule, e.g., an scFv domain, that binds CTLA4 expressed on the surface of a Treg. The therapeutic molecule accumulates or retains CTLA4+ tregs at the target site, with local immunosuppression as a result.

Although expressed higher on tregs, CTLA4 is also expressed on activated T cells. Therapeutic compounds comprising effector binding/modulating moieties (e.g., anti-CTLA 4 antibodies or functional anti-CTLA 4 antibodies) can down-regulate CTLA 4-expressing T cells. Thus, in therapeutic compounds comprising an effector binding/modulating moiety that binds CTLA4, the effector moiety may also serve as an ICIM binding/modulating moiety.

In some embodiments, the anti-CTLA 4 binding agent is neither antagonistic nor agonistic when in monomeric form, and is only agonistic upon clustering or multimerization upon binding to a target.

While not wishing to be bound by theory, it is believed that the therapeutic compound effects multimerization of the therapeutic compound by binding of the targeting moiety to the target. In the case of memory and activated T cells, CTLA4 bound by the effector binding/regulatory portion of the therapeutic compound clusters and inhibitory signals are generated by engagement of CTLA4 expressed by memory and activated T cells.

In some embodiments, the anti-CTLA 4 binding agent is neither antagonistic nor agonistic when in monomeric form, and is only agonistic upon clustering or multimerization upon binding to a target.

GITR binding agents

GITR (CD357) is a cellular surface marker present on tregs. Blockade of GITR-GITRL interactions maintains Treg function. In some embodiments, the therapeutic compound comprises an IIC binding entity that binds to GITR-expressing cells, and a therapeutic compound that targets the therapeutic compound to a target tissue of interest.

In some embodiments, the therapeutic compound comprises an anti-GITR antibody molecule, e.g., an anti-GITR antibody molecule that inhibits GITR binding to GITRL.

In some embodiments, the therapeutic compound comprises an anti-GITR antibody molecule, an anti-GITR antibody molecule that inhibits GITR binding to GITRL, and a PD-1 agonist or other effector described herein.

While not wishing to be bound by theory, it is believed that therapeutic compounds comprising GITR binding agents effect accumulation of GITR-expressing tregs at the site targeted by the targeting moiety of the therapeutic compound (e.g., the site of graft or organ injury).

Cyctophilin/lactophilin-like molecules

The effector binding/modulating moiety may comprise an agonistic BTNL2 molecule. While not wishing to be bound by theory, it is believed that the agonistic BNL2 molecule induces Treg cells.

As the term is used herein, an agonistic BTNL2 molecule refers to a polypeptide with sufficient BTNL2 sequence to induce Treg cells as part of a therapeutic compound. In some embodiments, the BTNL2 molecule has at least 60, 70, 80, 90, 95, 99, or 100% sequence identity or substantial sequence identity to a naturally occurring cremophilic protein.

In some embodiments, the effector binding/modulating moiety comprises an antagonistic BTNL8 molecule.

Therapeutic compounds comprising an SM binding/modulating moiety: manipulation of local microenvironment

The therapeutic compound may comprise an effector binding/modulating moiety that promotes an immunosuppressive local microenvironment, for example by providing a substance proximal to the target that inhibits or minimizes the attack of the target's immune system (referred to herein as a SM binding/modulating moiety).

In some embodiments, the SM binding/modulating moiety comprises a molecule that inhibits or minimizes immune system attack of the target (referred to herein as the SM binding/modulating moiety). In some embodiments, the therapeutic compound comprises an SM binding/modulating moiety that binds to and accumulates soluble substances with immunosuppressive functions, such as endogenous or exogenous substances. In some embodiments, the therapeutic compound comprises an SM binding/modulating moiety, such as a CD39 molecule or a CD73 molecule or an alkaline phosphatase molecule, that binds, inhibits, sequesters, degrades, or otherwise neutralizes soluble substances that promote immune attack, typically endogenous soluble substances, e.g., ATP in the case of a CD39 molecule or an alkaline phosphatase molecule; or AMP in the case of the CD73 molecule. In some embodiments, the therapeutic compound comprises an SM binding/modulating moiety comprising an immunosuppressive substance, e.g., a protein fragment that is immunosuppressive.

Donor tissue

The therapeutic compounds and methods described herein can be used in conjunction with transplantation of donor tissue into a subject, and can minimize rejection of the donor transplanted tissue; minimizing immune effector cell-mediated damage to donor transplant tissue; prolonged acceptance of donor graft tissue; or to extend the functional life of the donor graft tissue. The tissue may be a xenograft or an allograft tissue. The transplanted tissue may include all or part of an organ, such as the liver, kidney, heart, pancreas, thymus, skin, or lung. In embodiments, the therapeutic compounds described herein reduce or eliminate the need for systemic immunosuppression. The therapeutic compounds and methods described herein can also be used to treat GVHD. In some embodiments, the host cell is coated with a therapeutic compound comprising a PD-L1 molecule as an effector binding/modulating moiety.

Table 2 provides target molecules for transplantation indications. The target molecule is the target to which the targeting moiety binds. As discussed elsewhere herein, in some embodiments, a targeting moiety is selected that binds to the product of an allele present on the donor tissue and is not expressed by the subject (recipient) or is expressed at a different level (e.g., a reduced or significantly reduced level).

Autoimmune disorders

The therapeutic compounds and methods described herein can be used to treat subjects having or at risk of having an undesired autoimmune response, such as type 1 diabetes, multiple sclerosis, myocarditis, vitiligo, alopecia, inflammatory bowel disease (IBD, e.g., crohn's disease or ulcerative colitis), Sjogren's syndrome, Focal Segmental Glomerulosclerosis (FSGS), scleroderma/systemic sclerosis (SSc), or rheumatoid arthritis. In some embodiments, the treatment minimizes rejection of a tissue of the subject that is or is at risk of experiencing an immune attack, minimizes immune effector cell-mediated damage to the tissue of the subject, or prolongs the survival of the tissue of the subject. Table 3 provides target molecules for several autoimmune indications and organ/cell types. The target molecule is the target to which the targeting moiety binds.

Other examples of autoimmune conditions and diseases that can be treated with the compounds described herein include, but are not limited to, myocarditis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, subacute bacterial endocarditis, anti-glomerulobasement 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, pemphigoid gestationis, hidrosis suppurativa, lichen planus, lichen sclerosus, linear iga disease (lad), Maculopathy, pemphigus vulgaris, acute lichen pityriasis rubra, muckle-haydia (mucha-habermann disease), psoriasis, systemic scleroderma, vitiligo, adison's disease, autoimmune multiple endocrine syndrome (APS) type 1, autoimmune multiple endocrine syndrome (APS) type 2, autoimmune multiple endocrine syndrome (APS) type 3, autoimmune pancreatitis (AIP), type 1 diabetes, autoimmune thyroiditis, alder's thyroiditis, Graves ' disease, autoimmune oophoritis, endometriosis, autoimmune orchitis, sjogren's syndrome, autoimmune enteropathy, celiac disease, crohn's disease, microscopic colitis, ulcerative colitis, thrombocytopenia, obesity, paresthesia, Adult Still's disease (Adult-onel's disease), Ankylosing spondylitis, CREST syndrome, drug-induced lupus, arthritis associated with attachment point inflammation, eosinophilic fasciitis, fischer syndrome (Felty syndrome), IgG 4-related diseases, juvenile arthritis, Lyme disease (chronic), Mixed Connective Tissue Disease (MCTD), recurrent rheumatism, Parry Romberg syndrome, Parsonage-Turner syndrome, psoriatic arthritis, reactive arthritis, recurrent polychondritis, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schnitzler syndrome, Systemic Lupus Erythematosus (SLE), Undifferentiated Connective Tissue Disease (UCTD), dermatomyositis, fibromyalgia, inclusion body myositis, myasthenia gravis, neuromuscular ankylosis, paraneoplastic cerebellar degeneration, polymyositis, Acute Disseminated Encephalomyelitis (ADEM), acute motor axis neuropathy, neuronopathy, anti-N-methyl D-aspartate (anti-NMDA) receptor encephalitis, Barlow's concentric sclerosis, Bickerstaff encephalitis, chronic inflammatory demyelinating polyneuropathy, Guillain-Barre syndrome, Hashimoto's encephalopathetic disease, idiopathic inflammatory demyelinating disease, Lambert-Eamastic syndrome, multiple sclerosis, Oshtora syndrome, Streptococcus-related autoimmune neuropsychiatric disorders (PANDAS), progressive inflammatory neuropathy, restless leg syndrome, stiff person syndrome, West Denham chorea (Sydenham chorea), transverse myelitis, autoimmune retinopathy, autoimmune uveitis, Cogan syndrome (Cogan syndrome), Graves ophthalmopathy (Graves ophtalopathy), intermediate conjunctivitis, keratoderma ulcer, bombyx mori ulcer, and silkworm ulcer, Neuromyelitis optica, ocular-myoclonus syndrome, optic neuritis, scleritis, Susac syndrome, sympathetic ophthalmia, Toloxa-Hunter syndrome, Autoimmune Inner Ear Disease (AIED), Meniere's disease, Behcet's disease, Eosinophilic Granulomatous Polyangiitis (EGPA), giant cell arteritis, Granulomatous Polyangiitis (GPA), IgA vasculitis (IgAV), Kawasaki's disease, leukocyte disruptive vasculitis, lupus vasculitis, rheumatoid vasculitis, Microscopic Polyangiitis (MPA), polyarteritis nodosa (PAN), polymyalgia rheumatica, vasculitis, primary immunodeficiency, and the like.

Other examples of potential autoimmune disorders and diseases and autoimmune complications that may be treated with the compounds described herein include, but are not limited to, chronic fatigue syndrome, complex regional pain syndrome, eosinophilic esophagitis, gastritis, interstitial lung disease, POEMS syndrome, Raynaud's phenomenon, primary immunodeficiency, pyoderma gangrenosum, agammaglobulinemia, amyloidosis, amyotrophic lateral sclerosis, anti-tubular basement membrane nephritis, atopic allergy, atopic dermatitis, autoimmune peripheral neuropathy, Blau syndrome, Castleman's disease, Coniosis americana, chronic obstructive pulmonary disease, chronic relapsing multifocal osteomyelitis, complement component 2 deficiency, contact dermatitis, Cushing's syndrome, cutaneous leukocytic vasculitis, Dego's disease, eczema, eosinophilic gastroenteritis, chronic relapsing myelogenous leukemia, chronic inflammatory bowel disease, chronic fatigue syndrome, chronic inflammatory bowel disease, chronic fatigue syndrome, eosinophilic pneumonia, fetal erythrocytosis, progressive fibrodysplasia ossificans, gastrointestinal pemphigoid, hypogammaglobulinemia, idiopathic giant cell myocarditis, idiopathic pulmonary fibrosis, IgA nephropathy, immunomodulatory lipoproteins, IPEX syndrome, woody conjunctivitis, Majeed syndrome, narcolepsy, Rasmussen encephalitis, schizophrenia, seropathy, spondyloarthritis, scurver's syndrome, Takayasu's arteritis, and the like.

In some embodiments, the autoimmune disorder does not include pemphigus vulgaris, pemphigus. In some embodiments, the autoimmune disorder does not include pemphigus foliaceus. In some embodiments, the autoimmune disorder does not include bullous pemphigoid. In some embodiments, the autoimmune disorder does not include Goodpasture's disease. In some embodiments, the autoimmune disorder does not include psoriasis. In some embodiments, the autoimmune disorder does not include a skin disorder. In some embodiments, the disorder does not include a neoplastic disorder, such as cancer.

Therapeutic compounds

The therapeutic compounds include a specific targeting moiety functionally associated with an effector binding/modulating moiety. In some embodiments, the specific targeting moiety and the effector binding/modulating moiety are linked to each other by covalent or non-covalent bonds, e.g., covalent or non-covalent bonds directly linking each other. In other embodiments, the specific targeting moiety and the effector binding/modulating moiety are linked by a linker moiety (e.g., covalent or non-covalent). For example, in the case of fusion polypeptides, the polypeptide sequence comprising the specific targeting moiety and the polypeptide sequence may be linked to each other directly or through one or more linker sequences. In some embodiments, the linker moiety comprises a polypeptide. However, the linker is not limited to a polypeptide. In some embodiments, the linker moiety comprises an additional backbone, such as a non-peptidic polymer, e.g., a PEG polymer. In some embodiments, the linker moiety may comprise a particle, such as a nanoparticle, e.g., a polymeric nanoparticle. In some embodiments, the linker moiety may comprise a branched molecule or dendrimer. However, in embodiments where the effector binding/modulating moiety comprises an ICIM binding/modulating moiety (which binds an effector, such as PD-1), 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. Thus, in embodiments, the therapeutic compound causes the structure (e.g., copies of the ICIM) to be sufficiently limited such that clustering is minimized or substantially eliminated or eliminated in the absence of target binding, or sufficiently minimized such that significant systemic immunosuppression does not occur.

In some embodiments, the therapeutic compound comprises a polypeptide comprising a specific targeting moiety covalently or non-covalently bound to an effector binding/modulating moiety. In some embodiments, the therapeutic molecule comprises a fusion protein comprising a specific targeting moiety fused to an effector binding/modulating moiety, e.g., directly or through a linking moiety comprising one or more amino acid residues. In some embodiments, the therapeutic molecule comprises a polypeptide comprising a specific targeting moiety linked to an effector binding/modulating moiety by a non-covalent bond or a covalent bond (e.g., a covalent bond other than a peptide bond, such as a sulfhydryl bond).

In some embodiments, the therapeutic compound comprises a polypeptide, e.g., a fusion polypeptide, comprising:

1.a) a specific targeting moiety comprising a target specific binding polypeptide;

1.b) a specific targeting moiety comprising a target ligand binding molecule;

1.c) a specific targeting moiety comprising an antibody molecule;

1.d) a specific targeting moiety comprising a single chain antibody molecule (e.g. a scFv domain); or

E) a specific targeting moiety comprising a first light or heavy chain variable region of an antibody molecule, and wherein the other variable region is covalently or non-covalently bound to the first variable region;

and

a) an effector binding/modulating moiety comprising an effector specific binding polypeptide;

2.b) an effector binding/modulating moiety comprising an effector ligand binding molecule;

2.c) an effector binding/modulating moiety comprising an antibody molecule;

2.d) an effector binding/modulating moiety comprising a single chain antibody molecule (e.g. a scFv domain); or

E) an effector binding/modulating portion comprising a first light or heavy chain variable region of an antibody molecule, and wherein the other variable region is covalently or non-covalently bound to the first variable region.

In some embodiments, the therapeutic compound comprises 1.a and 2. a.

In some embodiments, the therapeutic compound comprises 1.a and 2. b.

In some embodiments, the therapeutic compound comprises 1.a and 2. c.

In some embodiments, the therapeutic compound comprises 1.a and 2. d.

In some embodiments, the therapeutic compound comprises 1.a and 2. e.

In some embodiments, the therapeutic compound comprises 1.b and 2. a.

In some embodiments, the therapeutic compound comprises 1.b and 2. b.

In some embodiments, the therapeutic compound comprises 1.b and 2. c.

In some embodiments, the therapeutic compound comprises 1.b and 2. d.

In some embodiments, the therapeutic compound comprises 1.b and 2. e.

In some embodiments, the therapeutic compound comprises 1.c and 2. a.

In some embodiments, the therapeutic compound comprises 1.c and 2. b.

In some embodiments, the therapeutic compound comprises 1.c and 2. c.

In some embodiments, the therapeutic compound comprises 1.c and 2. d.

In some embodiments, the therapeutic compound comprises 1.c and 2. e.

In some embodiments, the therapeutic compound comprises 1.d and 2. a.

In some embodiments, the therapeutic compound comprises 1.d and 2. b.

In some embodiments, the therapeutic compound comprises 1.d and 2. c.

In some embodiments, the therapeutic compound comprises 1.d and 2. d.

In some embodiments, the therapeutic compound comprises 1.d and 2. e.

In some embodiments, the therapeutic compound comprises 1.e and 2. a.

In some embodiments, the therapeutic compound comprises 1.e and 2. b.

In some embodiments, the therapeutic compound comprises 1.e and 2. c.

In some embodiments, the therapeutic compound comprises 1.e and 2. d.

In some embodiments, the therapeutic compound comprises 1.e and 2. e.

The therapeutic compounds disclosed herein may, for example, comprise multiple effector binding/modulating and specific targeting moieties. The multiple portions may be presented using any suitable connectors or platforms. The linker is typically coupled to or fused to one or more effector binding/modulating and targeting moieties.

In some embodiments, two (or more) linkers are associated covalently or non-covalently, e.g., to form a hetero-or homodimeric therapeutic compound. For example, a linker may comprise an Fc region and the two Fc regions are associated with each other. In some embodiments of therapeutic compounds comprising two linker regions, the linker regions may self-associate, e.g., two identical Fc regions. In some embodiments of therapeutic compounds comprising two linker regions, the linker regions are not or not significantly self-associated, e.g., the two Fc regions can be members of a knob-hole pair.

Non-limiting exemplary configurations of therapeutic compounds include the following (e.g., in N-to C-terminal order):

r1- -linker region A- -R2

R3- -linker region B- -R4

Wherein the content of the first and second substances,

r1, R2, R3 and R4 each independently comprise an effector binding/modulating moiety, such as an ICIM binding/modulating moiety, an IIC 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 each other, e.g., linker a and linker B each comprise an Fc moiety, provided that an effector binding/modulating moiety and a specific targeting moiety are present.

In some embodiments:

r1 comprises an effector binding/modulating moiety, such as an ICIM binding/modulating moiety, an IIC 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, such as an ICIM binding/modulating moiety, an IIC 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 each other, e.g., linker a and linker B each comprise an Fc moiety, provided that one of R1 or R3 is present and one of R2 or R4 is present.

In some embodiments:

r1 comprises a specific targeting moiety, or is absent;

r2 comprises an effector binding/modulating moiety, such as an ICIM binding/modulating moiety, an IIC 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, such as an ICIM binding/modulating moiety, an IIC binding/modulating moiety, or an SM binding/modulating moiety, or is absent;

linker region a and linker B comprise moieties that can associate with each other, e.g., linker a and linker B each comprise an Fc moiety, provided that one of R1 or R3 is present and one of R2 or R4 is present.

Non-limiting examples include, but are not limited to:

in some embodiments:

r1, R2, R3 and R4 each independently comprise: an effector binding regulatory moiety that activates an inhibitory receptor on an immune cell (e.g., a T cell or B cell), such as a PD-L1 molecule or a functional anti-PD-1 antibody molecule (an agonist of PD-1); a specific targeting moiety; or is absent;

provided that an effector binding moiety and a specific targeting moiety are present.

In some embodiments, linker a and linker B comprise an Fc portion (e.g., a self-pairing Fc portion).

In some embodiments:

r1 and R3 independently comprise: an effector binding regulatory moiety that activates an inhibitory receptor on an immune cell (e.g., a T cell or B cell), such as a PD-L1 molecule or a functional anti-PD-1 antibody molecule (an agonist of PD-1); and is

R2 and R4 independently comprise specific targeting moieties, such as scFv molecules against a tissue antigen.

In some embodiments, linker a and linker B comprise an Fc portion (e.g., a self-pairing Fc portion).

In some embodiments:

r1 and R3 independently comprise a functional anti-PD-1 antibody molecule (agonist of PD-1); and is

R2 and R4 independently comprise specific targeting moieties, such as scFv molecules against a tissue antigen.

In some embodiments, linker a and linker B comprise an Fc portion (e.g., a self-pairing Fc portion).

In some embodiments:

r1 and R3 independently comprise a specific targeting moiety, such as an anti-tissue antigen antibody; and is

R2 and R4 independently comprise functional anti-PD-1 antibody molecules (agonists of PD-1), such as scFv molecules.

In some embodiments, linker a and linker B comprise an Fc portion (e.g., a self-pairing Fc portion).

In some embodiments:

r1 and R3 independently comprise a PD-L1 molecule (an agonist of PD-1); and is

R2 and R4 independently comprise a specific targeting moiety, such as an scFv molecule against a tissue antigen; and is

In some embodiments, linker a and linker B comprise an Fc portion (e.g., a self-pairing Fc portion).

In some embodiments:

r1 and R3 independently comprise a specific targeting moiety, such as an anti-tissue antigen antibody; and is

R2 and R4 independently comprise a PD-L1 molecule (an agonist of PD-1).

In some embodiments, linker a and linker B comprise an Fc portion (e.g., a self-pairing Fc portion).

In some embodiments:

r1, R2, R3 and R4 each independently comprise: SM binding/modulating moieties that modulate, e.g., bind and inhibit, sequester, degrade, or otherwise neutralize 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;

provided that an SM binding/modulating moiety and a specific targeting moiety are present.

In some embodiments, linker a and linker B comprise Fc portions (e.g., self-pairing Fc portions or Fc portions that do not self-pair or substantially do not self-pair).

In some embodiments:

r1 and R3 each independently comprise: SM binding/modulating moieties that modulate, e.g., bind and inhibit, sequester, degrade, or otherwise neutralize 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 is

R2 and R4 independently comprise specific targeting moieties, such as scFv molecules against a tissue antigen.

In some embodiments, linker a and linker B comprise Fc portions (e.g., self-pairing Fc portions or Fc portions that do not self-pair or substantially do not self-pair).

In some embodiments:

r1 and R3 independently comprise a CD39 molecule or a CD73 molecule; and is

R2 and R4 independently comprise specific targeting moieties, such as scFv molecules against a tissue antigen.

In some embodiments, linker a and linker B comprise Fc portions (e.g., self-pairing Fc portions or Fc portions that do not self-pair or substantially do not self-pair).

In some embodiments:

r1 and R3 each comprise a CD39 molecule; and is

R2 and R4 independently comprise a specific targeting moiety, such as an scFv molecule against a tissue antigen; and is

In some embodiments, linker a and linker B comprise Fc portions (e.g., self-pairing Fc portions or Fc portions that do not self-pair or substantially do not self-pair).

In some embodiments:

r1 and R3 each comprise a CD73 molecule; and is

R2 and R4 independently comprise specific targeting moieties, such as scFv molecules against a tissue antigen.

In some embodiments, linker a and linker B comprise Fc portions (e.g., self-pairing Fc portions or Fc portions that do not self-pair or substantially do not self-pair).

In some embodiments:

one of R1 and R3 comprises a CD39 molecule and the other comprises a CD73 molecule; and is

R2 and R4 independently comprise specific targeting moieties, such as scFv molecules against a tissue antigen.

In some embodiments, linker a and linker B comprise Fc portions (e.g., self-pairing Fc portions or Fc portions that do not self-pair or substantially do not self-pair).

In some embodiments:

r1, R2, R3 and R4 each independently comprise: an HLA-G molecule; a specific targeting moiety; or is absent;

provided that an HLA-G molecule and a specific targeting moiety are present.

In some embodiments, linker a and linker B comprise Fc portions (e.g., self-pairing Fc portions or Fc portions that do not self-pair or substantially do not self-pair).

In some embodiments:

r1 and R3 each comprise a HLG-a molecule; and is

R2 and R4 independently comprise specific targeting moieties, such as scFv molecules against a tissue antigen.

In some embodiments, linker a and linker B comprise Fc portions (e.g., self-pairing Fc portions or Fc portions that do not self-pair or substantially do not self-pair).

In some embodiments:

r1 and R3 each comprise an antagonist anti-LILRB 1 antibody molecule; and is

R2 and R4 independently comprise specific targeting moieties, such as scFv molecules against a tissue antigen.

In some embodiments, linker a and linker B comprise Fc portions (e.g., self-pairing Fc portions or Fc portions that do not self-pair or substantially do not self-pair).

In some embodiments:

r1 and R3 each comprise an antagonist anti-KIR 2DL4 antibody molecule; and is

R2 and R4 independently comprise specific targeting moieties, such as scFv molecules against a tissue antigen.

In some embodiments, linker a and linker B comprise Fc portions (e.g., self-pairing Fc portions or Fc portions that do not self-pair or substantially do not self-pair).

In some embodiments:

r1 and R3 each comprise an antagonist anti-LILRB 2 antibody molecule; and is

R2 and R4 independently comprise specific targeting moieties, such as scFv molecules against a tissue antigen.

In some embodiments, linker a and linker B comprise Fc portions (e.g., self-pairing Fc portions or Fc portions that do not self-pair or substantially do not self-pair).

In some embodiments:

r1 and R3 each comprise an antagonist anti-NKG 2A antibody molecule; and is

R2 and R4 independently comprise specific targeting moieties, such as scFv molecules against a tissue antigen.

In some embodiments, linker a and linker B comprise Fc portions (e.g., self-pairing Fc portions or Fc portions that do not self-pair or substantially do not self-pair).

In some embodiments:

one of R1 and R3 comprises a first moiety selected from the group consisting of: antagonist anti-LILRB 1 antibody molecules, agonist anti-KR 2DL4 antibody molecules, and agonist anti-NKG 2A antibody molecules; and is

R2 and R4 independently comprise specific targeting moieties, such as scFv molecules against a tissue antigen.

In some embodiments, linker a and linker B comprise Fc portions (e.g., self-pairing Fc portions or Fc portions that do not self-pair or substantially do not self-pair).

In some embodiments:

one of R1 and R3 comprises an antagonist anti-LILRB 1 antibody molecule and the other comprises an agonistic anti-KR 2DL4 antibody molecule; and is

R2 and R4 independently comprise specific targeting moieties, such as scFv molecules against a tissue antigen.

In some embodiments, linker a and linker B comprise Fc portions (e.g., self-pairing Fc portions or Fc portions that do not self-pair or substantially do not self-pair).

In some embodiments:

one of R1 and R3 comprises an antagonist anti-LILRB 1 antibody molecule and the other comprises an agonistic anti-NKG 2A antibody molecule; and is

R2 and R4 independently comprise specific targeting moieties, such as scFv molecules against a tissue antigen.

In some embodiments, linker a and linker B comprise Fc portions (e.g., self-pairing Fc portions or Fc portions that do not self-pair or substantially do not self-pair).

In some embodiments:

r1, 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;

provided that an effector binding moiety and a specific targeting moiety are present.

In some embodiments, linker a and linker B comprise Fc portions (e.g., self-pairing Fc portions or Fc portions that do not self-pair or substantially do not self-pair).

In embodiments, 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 FCRL 6.

In some embodiments:

r1 and R3 each comprise an antagonist anti-FCRL antibody molecule; and is

R2 and R4 independently comprise specific targeting moieties, such as scFv molecules against a tissue antigen.

In some embodiments, linker a and linker B comprise Fc portions (e.g., self-pairing Fc portions or Fc portions that do not self-pair or substantially do not self-pair).

In embodiments, 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 FCRL 6.

In some embodiments:

r1 and R3 independently comprise a specific targeting moiety, such as an antibody against a tissue antigen; and is

R2 and R4 each comprise an anti-FCRL antibody molecule, e.g., an agonistic anti-FCRL antibody molecule, e.g., an scFv molecule.

In some embodiments, linker a and linker B comprise Fc portions (e.g., self-pairing Fc portions or Fc portions that do not self-pair or substantially do not self-pair).

In embodiments, 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 FCRL 6.

In some embodiments:

one of R1, R2, R3, and R4 comprises an anti-BCR antibody molecule, e.g., an antagonist anti-BCR antibody molecule, one comprises an anti-FCRL antibody molecule, and one comprises a specific targeting moiety.

In some embodiments, linker a and linker B comprise Fc portions (e.g., self-pairing Fc portions or Fc portions that do not self-pair or substantially do not self-pair).

In some embodiments, 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 FCRL 6.

In some embodiments:

one of R1, R2, R3, and R4 comprises a bispecific antibody molecule comprising an anti-BCR antibody molecule, e.g., an antagonist anti-BCR antibody molecule, and an anti-FCRL antibody molecule, and one comprises a specific targeting moiety;

in some embodiments, linker a and linker B comprise Fc portions (e.g., self-pairing Fc portions or Fc portions that do not self-pair or substantially do not self-pair).

In embodiments, 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 FCRL 6.

In some embodiments:

r1, R2, R3 and R4 each independently comprise:

i) an effector binding/modulating moiety, such as an ICIM binding/modulating moiety, an IIC binding/modulating moiety, an ICSM binding/modulating moiety or an SM binding/modulating moiety, which minimizes or inhibits T cell activity, expansion or function (T cell effector binding/modulating moiety);

ii) an effector binding/modulating moiety, such as an ICIM binding/modulating moiety, an IIC binding/modulating moiety, an ICSM binding/modulating moiety or an SM binding/modulating moiety, which minimizes or inhibits B cell activity, expansion or function (B cell effector binding/modulating moiety);

iii) a specific targeting moiety; or

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.

In some embodiments, linker a and linker B comprise an Fc portion (e.g., a self-pairing Fc portion).

In some embodiments, one of R1, R2, R3, and R4 comprises an agonistic anti-PD-1 antibody, and one comprises an HLA-G molecule.

In some embodiments, one of R1, R2, R3, and R4 comprises an SM binding/modulating moiety, such as a CD39 molecule or a CD73 molecule. In some embodiments, one of R1, R2, R3, and R4 comprises an entity that binds to, activates, or maintains a regulatory immune cell (e.g., a Treg cell or Breg cell).

In some embodiments, one of R1, R2, R3, and R4 comprises an agonistic anti-PD-1 antibody, or one comprises an HLA-G molecule. In some embodiments, one of R1, R2, R3, and R4 comprises an agonistic anti-PD-1 antibody, one comprises an HLA-G molecule, and one comprises a CD39 molecule or a CD73 molecule.

Connecting sub-regions

As discussed elsewhere herein, the specific targeting and effector binding/modulating moieties may be linked by a linker region. Any of the linker regions described herein may be used as a linker. For example, linker regions a and B may comprise an Fc region. In some embodiments, the therapeutic compound comprises a linker region that can self-associate. In some embodiments, the therapeutic compound comprises a linker region having a moiety that minimizes self-association, and typically linker region a and linker region B are heterodimers. Linkers also include glycine/serine linkers. In some embodiments, the linker may comprise one or more repeats of GGGGS (SEQ ID NO: 6). In some embodiments, the linker comprises 1, 2, 3, 4, or 5 repeats. In some embodiments, the linker comprises GGGGSGGGGS (SEQ ID NO: 7). In some embodiments, the linker comprises GGGGSGGGGSGGGS (SEQ ID NO: 8). These linkers can be used in any of the therapeutic compounds or compositions provided herein.

In some embodiments, a therapeutic compound comprises, wherein the therapeutic compound is a polypeptide. In some embodiments, the polypeptide comprises an antibody at the N-terminus, the antibody consisting of F (ab ')2 on the IgG1Fc backbone, the F (ab')2 fused to an scFv at the C-terminus of an IgG Fc backbone. In some embodiments, the IgG Fc scaffold is an IgG1Fc scaffold. In some embodiments, the IgG1 backbone is substituted with an IgG4 backbone, an IgG2 backbone, or other similar IgG backbone. The IgG scaffold described in this paragraph can be used throughout this application, where the Fc region is referred to as part of the therapeutic compound. Thus, in some embodiments, the antibody consisting of F (ab')2 on the IgG1Fc backbone can be an anti-MAdCAM antibody or an anti-PD-1 antibody on IgG1Fc or any other targeting or effector binding/modulating moiety provided herein. In some embodiments, the scFV fragment fused to the C-terminus can be an anti-PD-1 antibody when the N-terminal region is an anti-MAdCAM antibody, or an anti-MAdCAM antibody when the N-terminal region is an anti-PD-1 antibody. In this non-limiting example, the N-terminus can be a targeting moiety, such as any of the targeting moieties provided herein, and the C-terminus can be an effector binding/modulating moiety, such as any of the effector binding/modulating moieties provided herein. Alternatively, in some embodiments, the N-terminus can be an effector binding/modulating moiety, such as any of the effector binding/modulating moieties provided herein, and the C-terminus can be a targeting moiety, such as any of the targeting moieties provided herein.

In some embodiments, the N-terminus can be a targeting moiety, such as any of the targeting moieties provided herein, and the C-terminus can be an effector binding/modulating moiety, such as any of the effector binding/modulating moieties provided herein.

In some embodiments, the therapeutic compound comprises two homodimeric polypeptides. In some embodiments, the N-terminus of the polypeptide comprises an effector binding/modulating moiety fused to a human IgG1Fc domain (e.g., a CH2 and/or CH3 domain). In some embodiments, the C-terminus of the Fc domain is another linker fused to the targeting moiety. Thus, in some embodiments, the molecule may be represented using the formula R1-linker a-Fc region-linker B-R2, wherein R1 may be an effector binding/modulating moiety, R2 is a targeting moiety, and linker a and linker B are independently linkers as provided herein. In some embodiments, linker 1 and linker 2 are different.

In some embodiments, the molecule may be represented using the formula R1-linker a-Fc region-linker B-R2, where R1 may be the targeting moiety, R2 is the effector binding/modulating moiety, and linker a and linker B are independently linkers as provided herein. In some embodiments, linker a and linker B are different. The linker may be selected from the non-limiting examples provided herein. In some embodiments, R1 and R2 are independently selected from F (ab')2 and scFV antibody domains. In some embodiments, R1 and R2 are different antibody domains. In some embodiments, the scFV is in the VL-VH domain orientation.

In some embodiments, the therapeutic compound is a bispecific antibody. In some embodiments, the bispecific antibody is composed of four polypeptide chains comprising:

chain 1: nt-VH1-CH1-CH2-CH 3-linker A-scFv [ VL 2-linker B-VH2] -ct

Chain 2: nt-VH1-CH1-CH2-CH 3-linker A-scFv [ VL 2-linker B-VH2] -ct

Chain 3: nt-VL1-CL-ct

Chain 4: nt-VL1-CL-ct,

wherein chain 1 and chain 2 are identical to each other and chain 3 and chain 4 are identical to each other,

wherein chain 1 forms a homodimer with chain 2; and chain 3 and chain 4 associate with chain 1 and chain 2. That is, when each light chain associates with each heavy chain, VL1 associates with VH1 and CL associates with CH1 to form two functional Fab units. Without being bound by any particular theory, each scFv unit is functional in nature because VL2 and VH2 are linked in series with a linker as provided herein (e.g. GGGGSG (SEQ ID NO:6), GGGGSGGGGSGGGGGGSGGGGS (SEQ ID NO:9), GGGGSGGGGSGGGGS (SEQ ID NO:8) or GGGGSGGGGS (SEQ ID NO: 7). the sequence of linker A and linker B, independently of each other, may be the same or different and as further described throughout this application. thus, in some embodiments linker A comprises GGGGGGS (SEQ ID NO:6), GGSGGGGS (SEQ ID NO:7), GGGGGGGSGGGGGGGGGGGGGGGGS (SEQ ID NO:8) or GGGGSGGGGGGGGSGGGGGGSGGGGS (SEQ ID NO: 9). in some embodiments linker B comprises GGS (SEQ ID NO:6), GGGGGGGS (SEQ ID NO:7), GGGGGSGGGSGGGSGGGGGSGGGGGGGGSGGGGGGGGGSGGGS (SEQ ID NO:7) or GGGSGGGSGGGSGGGSGGGGGSGGSGGGSGGGNT-7-GGGSNT (SEQ ID NO: 369). GGGCT-7) or GGGSNT 369-GGGSNT 2 or GGGSNT-7-GGGSNO: 7-GGGSGGGSNO N-terminal and CT or CT represents the C-terminal of the protein. CH1, CH2, and CH3 are domains from the IgG Fc region, and CL represents a constant light chain, which may be a kappa or lambda family light chain. Other definitions represent the manner in which they are commonly used in the art.

In some embodiments, the VH1 and VL1 domains are derived from effector molecules, and the VH2 and VL2 domains are derived from targeting moieties. 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.

In some embodiments, the VH1 and VL1 domains are derived from an anti-PD-1 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-1 antibody.

In some embodiments, linker A comprises 1, 2, 3, 4, or 5 GGGGS (SEQ ID NO:6) repeats. In some embodiments, linker B comprises 1, 2, 3, 4, or 5 GGGGS repeats. For the avoidance of doubt, the sequences of linker a and linker B are used in this application independently of each other. Thus, in some embodiments, linker a and linker B may be the same or different. In some embodiments, linker A comprises GGGGS (SEQ ID NO:6), GGGGSGGGGS (SEQ ID NO:7), GGGGSGGGGSGGS (SEQ ID NO:8), or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 9). In some embodiments, linker B comprises GGGGS (SEQ ID NO:6), GGGGSGGGGS (SEQ ID NO:7), GGGGSGGGGSGGS (SEQ ID NO:8), or GGGGSGGGGSGGGGGS (SEQ ID NO: 9).

In some embodiments, the therapeutic compound comprises a light chain and a heavy chain. In some embodiments, the light and heavy chains start at the N-terminus with a VH domain of a targeting moiety, followed by a CH1 domain of human IgG1, which CH1 domain is fused to the Fc region of human IgG1 (e.g., CH2-CH 3). In some embodiments, the Fc region is fused at the c-terminus to a linker as provided herein, such as, but not limited to, GGGGS (SEQ ID NO:6), GGGGSGGGGS (SEQ ID NO:7), or ggggsggggsggs (SEQ ID NO: 8). The linker can then be fused to an effector binding/modulating moiety, such as any of the effector moieties provided herein. The polypeptide may homodimerize as a result of homodimerization by the heavy chain, which results in a therapeutic compound having two effector moieties (e.g., two anti-PD-1 antibodies). In this orientation, the targeting moiety is of the IgG format, there are two Fab arms, each of which recognizes a binding partner of the targeting moiety, e.g., MAdCAM bound to an anti-MAdCAM targeting moiety.

In some embodiments, if the therapeutic compound comprises an Fc portion, the Fc domain (portion) carries a mutation to "null" the Fc region that is unable to bind FcR. Mutations that null the Fc region are known. In some embodiments, the mutation in the Fc region (according to the known numbering system) is selected from the group consisting of: K322A, L235A, L236A, G237A, L235F, L236E, N297, P331S, or any combination thereof. In some embodiments, the Fc mutation comprises a mutation at L235 and/or L236 and/or G237. In some embodiments, the Fc mutation comprises an L235A and/or L236A mutation, which may be referred to as. In some embodiments, the Fc mutations comprise L235A, L236A, and G237A mutations.

Linker region polypeptides, therapeutic peptides, and nucleic acids encoding polypeptides (e.g., therapeutic compounds), vectors comprising nucleic acid sequences, and cells comprising nucleic acids or vectors are disclosed herein.

The therapeutic compound may comprise a plurality of specific targeting moieties. In some embodiments, the therapeutic compound comprises a plurality of one specific targeting moiety, a plurality of copies of a donor-specific targeting moiety, or a plurality of tissue-specific targeting moieties. In some embodiments, the therapeutic compound comprises first and second donor-specific targeting moieties, 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 targets are found on the same donor tissue. In some embodiments, the therapeutic compound comprises, for example, a first specific targeting moiety of a tissue-specific target and a second specific targeting moiety of a second target, e.g., wherein the first and second targets are present on the same or different target tissues.

In some embodiments, the therapeutic compound comprises a plurality of effector binding/modulating moieties, each moiety comprising an ICIM binding/modulating moiety, the number of ICIM binding/modulating moieties being sufficiently low such that clustering of ligands of the ICIM binding/modulating moieties on immune cells (in the absence of target binding) is minimized, e.g., to avoid systemic agonism of immune cells in the absence of binding of the therapeutic compound to the target.

Polypeptides derived from a reference, e.g. human polypeptides

In some embodiments, the component of the therapeutic molecule is derived from or based on a reference molecule, e.g., from a naturally occurring human polypeptide in the case of a therapeutic molecule for use in a human. For example, in some embodiments, all or part of a CD39 molecule, CD73 molecule, cell surface molecule binding agent, donor-specific targeting moiety, effector ligand binding molecule, ICIM binding/modulating moiety, IIC binding/modulating moiety, inhibitory immune checkpoint molecule ligand molecule, inhibitory molecule anti-ligand molecule, SM binding/modulating moiety, specific targeting moiety, target ligand binding molecule, or tissue-specific targeting moiety may be based on or derived from a naturally occurring human polypeptide. For example, the PD-L1 molecule can be based on or derived from a human PD-L1 sequence.

In some embodiments, the therapeutic compound component, e.g., PD-L1 molecule:

a) comprises all or part of a naturally occurring form of a human polypeptide, e.g., an active portion;

b) comprises all or part, e.g., an active portion, of a human polypeptide having a sequence that occurs in a database (e.g., a gene bank database, 1/11/2017, a naturally occurring form of a human polypeptide not associated with a disease state);

c) a human polypeptide comprising a sequence that differs from the sequence of a) or b) by no more than 1, 2, 3, 4, 5, 10, 20, or 30 amino acid residues;

d) a human polypeptide comprising a sequence having no more than 1, 2, 3, 4, 5, 10, 20 or 30% of its amino acid residues different from the sequence of a) or b);

e) a human polypeptide comprising a sequence substantially identical to the sequence of a) or b); or

f) A human polypeptide comprising a sequence of c), d) or e) having substantially the same biological activity (e.g., the ability to enhance or suppress an immune response) as a human polypeptide comprising a sequence of a) or b).

In some embodiments, a therapeutic compound may comprise multiple effector binding/modulating moieties. For example, the therapeutic compound may comprise two or more selected from:

(a) an ICIM binding/modulating moiety; (b) an IIC binding/modulating moiety; or (c) an SM binding/modulating moiety. In some embodiments, for example, a therapeutic compound may comprise a plurality, e.g., two, ICIM binding/modulating moieties (wherein they are the same or different); for example, two moieties that activate or agonize PD-1; a plurality, e.g., two IIC binding/modulating moieties; (wherein they are the same or different); or a plurality, e.g., two, SM binding/modulating moieties (wherein they are the same or different). In some embodiments, a therapeutic compound may comprise an ICIM binding/modulating moiety and an IIC binding/modulating moiety; an ICIM binding/modulating moiety and an SM binding/modulating moiety; IIC binding/modulating moiety and SM binding/modulating moiety. In some embodiments, the therapeutic compound comprises a plurality of targeting moieties. In some embodiments, the targeting moieties may be the same or different.

Pharmaceutical composition and kit

In another aspect, the embodiments of the invention provide a composition, e.g., a pharmaceutically acceptable composition, comprising a therapeutic compound described herein formulated with a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible.

The carrier may be suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, topical, ophthalmic, topical, spinal, or epidermal administration (e.g., by injection or infusion). The term "carrier" as used herein refers to a diluent, adjuvant, or excipient with which the compound is administered. In some embodiments, pharmaceutical carriers can also be liquids, such as water and oils, including those of petroleum, animal, vegetable, and synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Pharmaceutical carriers can also be saline, gum arabic, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, adjuvants, stabilizers, thickeners, lubricants and colorants may also be used. Carriers can be used in pharmaceutical compositions comprising the therapeutic compounds provided herein.

The compositions and compounds of the embodiments provided herein can 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. The preferred form depends on the intended mode of administration and therapeutic application. Typical compositions are in the form of injectable or infusible solutions. In some embodiments, the mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In some embodiments, the therapeutic molecule is administered by intravenous infusion or injection. In some embodiments, the therapeutic molecule is administered intramuscularly or by injection. In another embodiment, the therapeutic molecule is administered locally (e.g., by injection or topical coating) to the target site. As used herein, the phrases "parenteral administration" and "administered parenterally" mean modes of administration other than enteral and topical local administration, typically by injection, and include, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion.

The therapeutic compositions should generally be sterile and stable under the conditions of manufacture and storage. The compositions may be formulated as solutions, microemulsions, dispersions, liposomes or other ordered structures suitable for high therapeutic molecule concentrations. 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. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the other desired ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Proper solution fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin.

As the skilled person will appreciate, the route and/or manner of application will vary with the desired result. In certain embodiments, the active compound may be prepared with carriers that will protect the compound from rapid release, such as controlled release formulations, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid may be used. Many methods for preparing such formulations are patented or are generally known to those skilled in the art. See, e.g., Sustainated and controlled Release Drug Delivery Systems, ed.J.R. Robinson, Marcel Dekker, Inc., New York, 1978.

In certain embodiments, the therapeutic compound may be administered orally, for example, using an inert diluent or an assimilable edible carrier. The compounds (and optionally other ingredients) can also be enclosed in hard or soft shell gelatin capsules, compressed into tablets, or incorporated directly into the diet of a subject. For oral therapeutic administration, 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. In order to administer a compound by means other than parenteral administration, it may be necessary to coat the compound with a material, or to co-administer the compound with a material to prevent its deactivation. The therapeutic composition may also be administered with medical devices known in the art.

The dosage regimen is adjusted to provide the optimal desired response (e.g., 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 particularly advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. As used herein, dosage unit form refers to physically discrete units suitable as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the desired pharmaceutical carrier. The specifications for the dosage unit form are determined by and directly depend on the following factors: (a) the unique characteristics of the active compounds and the particular therapeutic effect to be achieved, and (b) limitations inherent in the art of compounding such active compounds for the treatment of sensitivity in individuals.

An exemplary, non-limiting range of therapeutically or prophylactically effective amount of a therapeutic compound is from 0.1 to 30mg/kg, more preferably from 1 to 25 mg/kg. The dosage of the therapeutic compound and the treatment regimen can be determined by the skilled artisan. In certain embodiments, the therapeutic compound is administered by injection (e.g., subcutaneously or intravenously) at a dose of about 1 to 40mg/kg, such as 1 to 30mg/kg, e.g., about 5 to 25mg/kg, about 10 to 20mg/kg, about 1 to 5mg/kg, 1 to 10mg/kg, 5 to 15mg/kg, 10 to 20mg/kg, 15 to 25mg/kg, or about 3 mg/kg. The dosing schedule may vary from, for example, once per week to once every 2, 3, or 4 weeks. In one embodiment, the therapeutic compound is administered at a dose of about 10 to 20mg/kg once every two weeks. The therapeutic compound may be administered by intravenous infusion at a rate in excess of 20mg/min, for example 20-40mg/min, and typically greater than or equal to 40mg/min, to achieve a dose of about 35 to 440mg/m2, typically about 70 to 310mg/m2, and more typically about 110 to 130mg/m 2. In embodiments, an infusion rate of about 110 to 130mg/m2 reaches a level of about 3 mg/kg. In other embodiments, the therapeutic compound may be administered by intravenous infusion at a rate of less than 10mg/min, such as less than or equal to 5mg/min, to achieve a dose of about 1 to 100mg/m2, such as about 5 to 50mg/m2, about 7 to 25mg/m2, or about 10mg/m 2. In some embodiments, the therapeutic compound is infused over a period of about 30 min. It should be noted that dosage values may vary with the type and severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the dosage ranges described herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions.

Pharmaceutical compositions can include a "therapeutically effective amount" or a "prophylactically effective amount" of a therapeutic molecule. By "therapeutically effective amount" is meant an amount effective, in dose and duration, to achieve the desired prophylactic result. The "therapeutically effective amount" of a therapeutic molecule can 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. A therapeutically effective amount is also a dose that has a therapeutically beneficial effect over any toxic or detrimental effects of the therapeutic molecule t. A "therapeutically effective dose" preferably inhibits a measurable parameter (e.g., immune challenge) by 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 an untreated subject. The ability of a compound to inhibit a measurable parameter (e.g., immune attack) can be assessed in an animal model system that predicts efficacy in transplant rejection or autoimmune disorders. Alternatively, such properties of the composition may be examined for the ability of the compound to inhibit such inhibition in vitro by assays known to the skilled artisan.

A "prophylactically effective amount" refers to an amount effective, in dosage and duration, to achieve the desired prophylactic result. Generally, since a prophylactic dose is used in a subject prior to or at an early stage of the disease, all prophylactically effective amounts will be less than therapeutically effective amounts.

Additionally, kits comprising the therapeutic compounds described herein are also within the scope of the embodiments. The kit may include one or more other elements including: instructions for use; other reagents, such as labels, therapeutic agents, or reagents for chelating or otherwise coupling a therapeutic molecule to a label or other therapeutic agent or radioprotective composition; devices or other materials for preparing therapeutic molecules for administration; a pharmaceutically acceptable carrier; and devices or other materials for administration to a subject.

In such embodiments, embodiments provided herein also include, but are not limited to:

1.a therapeutic compound comprising:

i) a specific targeting moiety selected from the group consisting of:

a) a donor-specific targeting moiety that, for example, preferentially binds to a donor target; or

b) A tissue-specific targeting moiety that, for example, preferentially binds to a target tissue of a subject; and

ii) an effector binding/modulating moiety selected from:

(a) an immune cell inhibitory molecule binding/modulating moiety (ICIM binding/modulating moiety);

(b) an immunosuppressive immune cell binding/modulating moiety (IIC binding/modulating moiety); or

(c) Effector binding/modulating moiety: it promotes an immunosuppressive local microenvironment (SM binding/modulating moiety) as part of a therapeutic compound, for example by providing a substance proximal to the target that inhibits or minimizes the attack of the target's immune system.

2. The therapeutic compound of embodiment 1 wherein the effector binding/modulating moiety directly binds to and activates an inhibitory receptor.

3. The therapeutic compound of embodiment 2, wherein the effector binding/modulating moiety is an inhibitory immune checkpoint molecule.

4. The therapeutic compound according to any one of embodiments 1 to 3, wherein the effector binding/modulating moiety is expressed by an immune cell.

5. The therapeutic compound of embodiment 4 wherein the immune cells promote an undesired immune response.

6. The therapeutic compound of embodiment 4 or 5, wherein the immune cells cause a disease condition.

7. The therapeutic compound of embodiment 1, wherein the ability of a therapeutic molecule to agonize binding/modulation of the bound molecule by the effector is greater, e.g., 2, 5, 10, 100, 500, or 1,000 times greater, when the therapeutic compound is bound to a target by the targeting moiety than the ability of a therapeutic molecule to agonize binding/modulation of the bound molecule by the effector when the therapeutic compound is not bound to a target by the targeting moiety.

8. The therapeutic compound according to embodiments 1 to 7, wherein the cognate ligand (e.g., an inhibitory immune checkpoint molecule) is not agonized or is not substantially agonized when bound as a monomer (or when the therapeutic compound is not multimerized) to its cognate ligand.

9. The therapeutic compound of embodiments 1 to 8 wherein there is significant systemic agonism of the molecule bound by the effector binding/modulating moiety at a therapeutically effective dose of the therapeutic compound.

10. The therapeutic compound according to embodiments 1 to 9, wherein agonism of the molecule bound by the effector binding/modulating moiety occurs substantially only at the target site bound by the targeting moiety at a therapeutically effective dose of the therapeutic compound.

11. The therapeutic compound according to embodiments 1 to 9, wherein binding of the therapeutic compound to its cognate ligand (e.g., an inhibitory immune checkpoint molecule) does not inhibit or does not substantially inhibit binding of an endogenous anti-ligand to the cognate ligand (e.g., an inhibitory immune checkpoint molecule).

12. The therapeutic compound according to embodiments 1 to 11 wherein binding of the effector binding/modulating moiety to its cognate ligand inhibits binding of the endogenous anti-ligand to the cognate ligand of the effector binding/modulating moiety by less than 60%, 50%, 40%, 30%, 20%, 10% or 5%.

14. The therapeutic compound of embodiments 1 through 11 wherein binding of the effector binding/modulating moiety to the cognate ligand has substantially no antagonistic effect on the cognate ligand of the effector binding/modulating molecule.

15. The therapeutic compound of embodiment 1, wherein the effector binding/modulating moiety comprises an ICIM binding/modulating moiety.

16. A therapeutic compound according to embodiment 15 wherein the effector binding/modulating moiety comprises an ICIM binding/modulating moiety comprising an inhibitory immune checkpoint molecule ligand molecule.

17. The therapeutic compound of embodiment 16, wherein the inhibitory immune molecule anti-ligand molecule comprises a PD-L1 molecule.

18. The therapeutic compound of embodiment 15, wherein the ICIM is wherein an inhibitory immune molecule anti-ligand molecule is conjugated to a cognate inhibitory immune checkpoint molecule selected from PD-1, KIR2DL4, LILRB1, LILRB, or CTLA-4.

19. The therapeutic compound of embodiment 18, wherein the ICIM is an antibody.

20. The therapeutic compound of embodiment 18, wherein the ICIM comprises an antibody that binds to PD-1, KIR2DL4, LILRB1, LILRB, or CTLA-4.

21. The therapeutic compound of embodiment 20, wherein the antibody is an antibody that binds to PD-1.

22. The therapeutic compound of embodiment 20 wherein the antibody is an antibody that binds to PD-1 and is a PD-1 agonist.

23. The therapeutic compound of embodiment 20, wherein the antibody is an antibody that binds to PD-1 and is a PD-1 agonist when tethered to a target site.

24. The therapeutic compound of embodiment 16, wherein the inhibitory immune molecule anti-ligand molecule comprises an HLA-G molecule.

25. The therapeutic compound of embodiment 15, wherein the ICIM is wherein an inhibitory immune molecule anti-ligand molecule is conjugated to a cognate inhibitory immune checkpoint molecule selected from PD-1, KIR2DL4, LILRB1, LILRB, or CTLA-4.

26. The therapeutic compound of embodiment 15 wherein the inhibitory immune molecule anti-ligand molecule engages a cognate inhibitory immune checkpoint molecule selected from table 1.

27. The therapeutic compound of embodiment 15 wherein the inhibitory immune checkpoint molecule is not agonized or is not substantially agonized when bound as a monomer to its cognate inhibitory immune checkpoint molecule.

28. The therapeutic compound of embodiment 15, wherein the inhibitory immune molecule anti-ligand has at least 60%, 70%, 80%, 90%, 95%, 99% or 100% homology to a naturally occurring inhibitory immune checkpoint molecule ligand.

29. The therapeutic compound of embodiment 1, wherein the effector binding/modulating moiety comprises an ICIM binding/modulating moiety comprising a functional antibody molecule to a cell surface inhibitory molecule.

30. The therapeutic compound of embodiment 1, wherein the cell surface inhibitory molecule is an inhibitory immune checkpoint molecule.

31. The compound of embodiment 30, wherein the inhibitory immune checkpoint molecule is selected from PD-1, KIR2DL4, LILRB1, LILRB2, CTLA-4, or from table 1.

32. The therapeutic compound according to any one of embodiments 1 to 31, wherein the level of systemic immunosuppression at a therapeutically effective dose of the therapeutic compound is lower than that provided by standard therapy with a systemic immunosuppressant (if relevant), or lower than that provided by an equimolar amount of free (not as a component of the therapeutic compound) effector binding/modulating molecule.

33. The therapeutic compound according to embodiments 1 to 32, wherein the systemic immune activation level is less than that provided by an equimolar amount of free (not as a component of the therapeutic compound) effector binding/modulating molecule, e.g. at a therapeutically effective dose of the therapeutic compound.

34. The therapeutic compound according to any one of embodiments 1-33, further comprising a second effector binding/modulating moiety.

35. The therapeutic compound of embodiment 34 wherein the second effector binding/modulating moiety binds to a different target than the effector binding/modulating moiety.

36. The therapeutic compound of embodiment 34 or 35, wherein the second effector binding/modulating moiety comprises an IIC binding/modulating moiety.

The therapeutic compound of embodiment 34 or 35, wherein the second effector binding/modulating moiety comprises an SM binding/modulating moiety.

37. The therapeutic compound of embodiment 1 wherein the effector binding/modulating moiety comprises an IIC binding/modulating moiety.

38. A therapeutic compound according to embodiment 1 wherein the effector binding/modulating moiety comprises an IIC binding/modulating moiety that increases, recruits or accumulates immunosuppressive immune cells at the target site.

39. The therapeutic compound of embodiment 1 wherein the effector binding/modulating moiety comprises a cell surface molecule binding agent that binds or specifically binds to a cell surface molecule on an immunosuppressive immune cell.

40. The therapeutic compound of embodiment 1 wherein the effector binding/modulating moiety comprises a cell surface molecule ligand molecule that binds or specifically binds to a cell surface molecule on an immunosuppressive immune cell.

41. The therapeutic compound of embodiment 1 wherein the effector binding/modulating moiety comprises an antibody molecule that binds to a cell surface molecule on an immunosuppressive immune cell.

42. The therapeutic compound of any one of embodiments 38-41, wherein immunosuppressive immune cells include T regulatory cells, such as Foxp3+ CD25+ T regulatory cells.

43. The therapeutic compound according to any one of embodiments 1 to 42, wherein the effector binding/modulating moiety binds to GARP and for example comprises an antibody molecule that binds to GARP on immunosuppressive cells (e.g. Tregs) that express GARP.

44. The therapeutic compound of embodiment 1 wherein the effector binding/modulating moiety comprises an SM binding/modulating moiety.

45. The therapeutic compound of embodiment 44 wherein the SM binding/modulating moiety promotes an immunosuppressive local microenvironment.

46. The therapeutic compound of any one of embodiments 44 and 45, wherein the effector molecule binding moiety increases availability, for example by increasing a local concentration or amount of a substance that inhibits immune cell function (e.g., a substance that inhibits immune cell activation or activates immune cell function).

47. The therapeutic compound of any one of embodiments 44-46 wherein the effector molecule binding moiety binds to and accumulates a soluble substance with immunosuppressive function, such as an endogenous or exogenous substance.

48. The therapeutic compound of any one of embodiments 44-47 wherein the effector molecule binding moiety reduces availability, for example by reducing the local concentration or amount of or sequestering a substance that promotes immune cell function (e.g., a substance that inhibits immune cell activation or activates immune cell function).

49. The therapeutic compound according to any one of embodiments 44-48, wherein the SM binding/modulating moiety promotes an immunosuppressive local microenvironment, for example by providing a substance proximal to the target that inhibits or minimizes the attack of the target's immune system.

50. The therapeutic compound according to any one of embodiments 44-49, wherein the SM binding/modulating moiety comprises a molecule that inhibits or minimizes the attack of the target immune system.

51. The therapeutic compound according to any one of embodiments 44 to 50, wherein the SM binding/modulating moiety binds to and/or accumulates a soluble substance with immunosuppressive function, such as an endogenous or exogenous substance.

52. The therapeutic compound according to any one of embodiments 44 to 51, wherein the SM binding/modulating moiety binds to and/or inhibits, sequesters, degrades or otherwise neutralizes a substance that promotes immune attack, typically an endogenous soluble molecule.

53. The therapeutic compound of any one of embodiments 44-52, wherein the effector molecule binding moiety reduces the availability of ATP or AMP.

54. The therapeutic compound according to any one of embodiments 44 to 53, wherein the SM binding/modulating moiety binds to or comprises a substance, such as CD39 or CD73, that depletes a component that promotes immune effector cell function, such as ATP or AMP.

55. The therapeutic compound according to any one of embodiments 44-54, wherein the SM binding/modulating moiety comprises a CD39 molecule.

56. The therapeutic compound according to any one of embodiments 44-54, wherein the SM binding/modulating moiety comprises a CD73 molecule.

57. The therapeutic compound according to any one of embodiments 44 to 54, wherein the SM binding/modulating moiety comprises an anti-CD 39 molecule.

58. The therapeutic compound according to any one of embodiments 44 to 54, wherein the SM binding/modulating portion comprises an anti-CD 73 antibody molecule.

59. The therapeutic compound of any one of embodiments 44-54 wherein the effector molecule binding moiety comprises an immunosuppressive substance, such as a fragment of an immunosuppressive protein.

60. The therapeutic compound according to any one of embodiments 44-54, wherein the SM binding/modulating moiety comprises an alkaline phosphatase molecule.

61. The therapeutic compound of embodiment 1, wherein the compound has the formula from N-terminus to C-terminus:

r1- -connecting sub-region A- -R2 or R3- -connecting sub-region B- -R4,

wherein the content of the first and second substances,

r1, R2, R3 and R4 each independently comprise an effector binding/modulating moiety, such as 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.

62. The therapeutic compound of embodiment 61, wherein each of linker region A and linker region B comprises an Fc region.

63. The therapeutic compound of embodiment 61 wherein one of R1 and R2 is an anti-PD-1 antibody and one of R1 and R2 is an anti-MAdCAM antibody.

64. The therapeutic compound of embodiment 61 wherein one R1 is an anti-PD-1 antibody and one R2 is an anti-MAdCAM antibody.

65. The therapeutic compound of embodiment 61 wherein one R1 is an anti-MAdCAM antibody and one R2 is an anti-PD-1 antibody.

66. The therapeutic compound of embodiment 61 wherein one of R3 and R4 is an anti-PD-1 antibody and one of R3 and R4 is an anti-MAdCAM antibody.

67. The therapeutic compound of embodiment 61 wherein one R3 is an anti-PD-1 antibody and one R4 is an anti-MAdCAM antibody.

68. The therapeutic compound of embodiment 61 wherein one R3 is an anti-MAdCAM antibody and one R4 is an anti-PD-1 antibody.

69. The therapeutic compound according to any one of embodiments 61-68, wherein a linker is absent.

70. The therapeutic compound of any one of embodiments 61-68, wherein the linker is an Fc region.

71. The therapeutic compound of any one of embodiments 61-68, wherein the linker is a glycine/serine linker, such as 1, 2, 3, 4, or 5 GGGGS (SEQ ID NO:6) repeats.

72. The therapeutic compound of any one of embodiments 61-68, wherein the linker comprises an Fc region and a glycine/serine linker, such as 1, 2, 3, 4, or 5 GGGGS (SEQ ID NO:6) repeats.

73. The therapeutic compound of any one of embodiments 61-72 wherein the PD-1 antibody is a PD-1 agonist.

74. The therapeutic compound of embodiment 61 wherein:

r1 and R3 independently comprise a functional anti-PD-1 antibody molecule (agonist of PD-1); and R2 and R4 independently comprise a specific targeting moiety, such as an scFv molecule directed against a tissue antigen.

75. The therapeutic compound according to any one of embodiments 73 and 74, wherein:

r1 and R3 independently comprise a specific targeting moiety, such as an anti-tissue antigen antibody; and R2 and R4 independently comprise a functional anti-PD-1 antibody molecule (an agonist of PD-1).

76. The therapeutic compound according to any one of embodiments 73 and 74, wherein:

r1, R2, R3 and R4 each independently comprise: SM binding/modulating moieties that modulate, e.g., bind and inhibit, sequester, degrade, or otherwise neutralize 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;

provided that an SM binding/modulating moiety and a specific targeting moiety are present.

77. The therapeutic compound of embodiment 61 wherein:

r1 and R3 independently comprise a CD39 molecule or a CD73 molecule; and is

R2 and R4 independently comprise specific targeting moieties, such as scFv molecules against a tissue antigen.

78. The therapeutic compound of embodiment 77 wherein:

r1 and R3 each comprise a CD39 molecule; and is

R2 and R4 independently comprise specific targeting moieties, such as scFv molecules against a tissue antigen.

79. The therapeutic compound of embodiment 61 or 77, wherein:

r1 and R3 each comprise a CD73 molecule; and is

R2 and R4 independently comprise specific targeting moieties, such as scFv molecules against a tissue antigen.

80. The therapeutic compound of embodiment 61 wherein:

one of R1 and R3 comprises a CD39 molecule and the other comprises a CD73 molecule; and is

R2 and R4 independently comprise specific targeting moieties, such as scFv molecules against a tissue antigen.

81. The therapeutic compound of embodiment 61 wherein:

r1, R2, R3 and R4 each independently comprise: an HLA-G molecule; a specific targeting moiety; or is absent;

provided that an HLA-G molecule and a specific targeting moiety are present.

82. The therapeutic compound of embodiment 61 or 81 wherein:

r1 and R3 each comprise a HLG-a molecule; and is

R2 and R4 independently comprise specific targeting moieties, such as scFv molecules against a tissue antigen.

In some embodiments, linker a and linker B comprise Fc portions (e.g., self-pairing Fc portions or Fc portions that do not self-pair or substantially do not self-pair).

83. The therapeutic compound according to any one of embodiments 81 and 82, wherein:

r1 and R3 each comprise an antagonist anti-LILRB 1 antibody molecule; and is

R2 and R4 independently comprise specific targeting moieties, such as scFv molecules against a tissue antigen.

In some embodiments, linker a and linker B comprise Fc portions (e.g., self-pairing Fc portions or Fc portions that do not self-pair or substantially do not self-pair).

84. The therapeutic compound according to any one of embodiments 81 and 82, wherein:

r1 and R3 each comprise an antagonist anti-KIR 2DL4 antibody molecule; and is

R2 and R4 independently comprise specific targeting moieties, such as scFv molecules against a tissue antigen.

In some embodiments, linker a and linker B comprise Fc portions (e.g., self-pairing Fc portions or Fc portions that do not self-pair or substantially do not self-pair).

85. The therapeutic compound according to any one of embodiments 81-84, wherein:

r1 and R3 each comprise an antagonist anti-LILRB 2 antibody molecule; and is

R2 and R4 independently comprise specific targeting moieties, such as scFv molecules against a tissue antigen.

86. The therapeutic compound according to any one of embodiments 81-84, wherein:

r1 and R3 each comprise an antagonist anti-NKG 2A antibody molecule; and is

R2 and R4 independently comprise specific targeting moieties, such as scFv molecules against a tissue antigen.

87. The therapeutic compound according to any one of embodiments 81-84, wherein:

one of R1 and R3 comprises a first moiety selected from the group consisting of: antagonist anti-LILRB 1 antibody molecules, agonist anti-KR 2DL4 antibody molecules, and agonist anti-NKG 2A antibody molecules; and is

R2 and R4 independently comprise specific targeting moieties, such as scFv molecules against a tissue antigen.

88. The therapeutic compound according to any one of embodiments 81-84, wherein:

one of R1 and R3 comprises an antagonist anti-LILRB 1 antibody molecule and the other comprises an agonistic anti-KR 2DL4 antibody molecule; and is

R2 and R4 independently comprise specific targeting moieties, such as scFv molecules against a tissue antigen.

89. The therapeutic compound according to any one of embodiments 81-84, wherein:

one of R1 and R3 comprises an antagonist anti-LILRB 1 antibody molecule and the other comprises an agonistic anti-NKG 2A antibody molecule; and is

R2 and R4 independently comprise specific targeting moieties, such as scFv molecules against a tissue antigen.

90. The therapeutic compound according to any one of embodiments 81-84, wherein:

one of R1, R2, R3, and R4 comprises an anti-BCR antibody molecule, e.g., an antagonist anti-BCR antibody molecule, one comprises an anti-FCRL antibody molecule, and one comprises a specific targeting moiety.

91. The therapeutic compound of embodiment 90 wherein:

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 FCRL 6.

92. The therapeutic compound according to any one of embodiments 81-84, wherein:

r1, R2, R3 and R4 each independently comprise:

i) an effector binding/modulating moiety, such as an ICIM binding/modulating moiety, an IIC binding/modulating moiety, or an SM binding/modulating moiety, which minimizes or inhibits T cell activity, expansion or function (T cell effector binding/modulating moiety);

ii) an effector binding/modulating moiety, such as an ICIM binding/modulating moiety, an IIC binding/modulating moiety, or an SM binding/modulating moiety, which minimizes or inhibits B cell activity, expansion or function (B cell effector binding/modulating moiety);

iii) a specific targeting moiety; or

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.

93. The therapeutic compound of embodiment 92 wherein:

one of R1, R2, R3, and R4 comprises an agonistic anti-PD-1 antibody, and one comprises an HLA-G molecule.

94. Therapeutic compound embodiments 92-93 wherein:

one of R1, R2, R3 and R4 comprises an SM binding/modulating moiety, such as a CD39 molecule or a CD73 molecule.

95. The therapeutic compound according to any one of embodiments 92-94, wherein:

one of R1, R2, R3, and R4 comprises an entity that binds to, activates, or maintains a regulatory immune cell (e.g., a Treg cell or Breg cell).

96. The therapeutic compound according to any one of embodiments 92-95, wherein:

one of R1, R2, R3, and R4 comprises an agonistic anti-PD-1 antibody, or one comprises an HLA-G molecule.

97. The therapeutic compound of embodiment 96 wherein:

one of R1, R2, R3, and R4 comprises an agonistic anti-PD-1 antibody, one comprises an HLA-G molecule, and one comprises a CD39 molecule or a CD73 molecule.

98. The therapeutic compound of any one of embodiments 1-97 wherein the effector binding/modulating moiety comprises a polypeptide.

99. The therapeutic compound of any one of embodiments 1-98, wherein the effector binding/modulating moiety comprises a polypeptide having at least 5, 10, 20, 30, 40, 50, 150, 200, or 250 amino acid residues.

100. The therapeutic compound according to any one of embodiments 1-99, wherein the molecular weight of the effector binding/modulating moiety is 5, 10, 15, 20 or 40 Kd.

101. The therapeutic compound according to any one of embodiments 1 to 100, wherein the effector binding/modulating moiety does not comprise an inhibitor of apolipoprotein CIII, protein kinase A, Src kinase or β 1 integrin expression.

102. The therapeutic compound according to any one of embodiments 1 to 100, wherein the effector binding/modulating moiety does not comprise an inhibitor of the activity of apolipoprotein CIII, protein kinase A, Src kinase or β 1 integrin.

103. The therapeutic compound of any one of embodiments 1-101, wherein the therapeutic compound does not specifically target a tissue selected from lung, skin, pancreas, retina, prostate, ovary, lymph node, adrenal gland, liver, or intestinal tissue.

104. The therapeutic compound of any one of embodiments 1-101, wherein the therapeutic compound does not specifically target tubular cells, e.g., proximal tubular epithelial cells.

105. The therapeutic compound according to any one of embodiments 1-101, wherein the therapeutic compound does not specifically target TIE-2, APN, TEM4, TEM6, ICAM-1, nucleolin P2Z receptor, Trk-A, FLJ10849, HSPA12B, APP, or OX-45.

106. The therapeutic compound of any one of embodiments 1-101, wherein the therapeutic compound does not specifically target a luminescently expressed protein.

107. The therapeutic compound of any one of embodiments 1-101, wherein donor target does not include a heart-specific target.

108. The therapeutic compound of any one of embodiments 1-101, wherein the therapeutic compound does not specifically target lung tissue.

109. The therapeutic compound of any one of embodiments 1-101, wherein the therapeutic compound does not specifically target kidney tissue.

110. The therapeutic compound of any one of embodiments 1-101, wherein the therapeutic compound does not specifically target pancreatic lung tissue.

111. The therapeutic compound of any one of embodiments 1-101, wherein the therapeutic compound does not specifically target intestinal tissue.

112. The therapeutic compound of any one of embodiments 1-101, wherein the therapeutic compound does not specifically target prostate tissue.

113. The therapeutic compound of any one of embodiments 1-101, wherein the therapeutic compound does not specifically target brain tissue.

114. The therapeutic compound of any one of embodiments 1-101, wherein the therapeutic compound does not specifically target CD 71.

115. The therapeutic compound of any one of embodiments 1-101, wherein the therapeutic compound does not specifically target CD 90.

116. The therapeutic compound of any one of embodiments 1-101, wherein the therapeutic compound does not specifically target MAdCAM.

117. The therapeutic compound of any one of embodiments 1-101, wherein the therapeutic compound does not specifically target albumin.

118. The therapeutic compound of any one of embodiments 1-101, wherein the therapeutic compound does not specifically target carbonic anhydrase IV.

119. The therapeutic compound of any one of embodiments 1-101, wherein the therapeutic compound does not specifically target ZG 16-p.

120. The therapeutic compound according to any one of embodiments 1-101, wherein the therapeutic compound does not specifically target dipeptidyl peptidase IV.

121. The therapeutic compound of any one of embodiments 1-101, wherein the therapeutic compound does not specifically target the luminal surface of the vascular endothelial cell membrane.

121. The therapeutic compound of any one of embodiments 1-101, wherein the therapeutic compound does not specifically target cardiac tissue.

122. The therapeutic compound of any one of embodiments 1-101, wherein the therapeutic compound does not specifically target a tumor, a solid tumor, or a blood vessel of a solid tumor.

123. The therapeutic compound of any one of embodiments 1-101, wherein the therapeutic compound does not specifically target skin tissue.

124. The therapeutic compound of any one of embodiments 1-101, wherein the therapeutic compound does not specifically target epidermal tissue.

125. The therapeutic compound of any one of embodiments 1-101, wherein the therapeutic compound does not specifically target basement membrane.

126. The therapeutic compound of any one of embodiments 1-101, wherein the therapeutic compound does not specifically target a Dsg polypeptide.

127. The therapeutic compound of any one of embodiments 1-101, wherein the therapeutic compound does not specifically target Dsg 1.

128. The therapeutic compound of any one of embodiments 1-101, wherein the therapeutic compound does not specifically target Dsg 3.

129. The therapeutic compound of any one of embodiments 1-101, wherein the therapeutic compound does not specifically target BP 180.

130. The therapeutic compound of any one of embodiments 1-101, wherein the therapeutic compound does not specifically target desmoglein.

131. The therapeutic compound according to any one of embodiments 1-101, wherein the therapeutic compound does not comprise a complement modulator, such as a complement inhibitor, for example, but not limited to, those described in U.S. patent No. 8,454,963, which is incorporated herein by reference in its entirety.

133. The therapeutic compound of any one of embodiments 1-101, wherein the therapeutic compound does not comprise an imaging agent.

134. The therapeutic compound according to any one of embodiments 1-101, wherein the therapeutic compound does not include an imaging agent selected from the group consisting of: radioactive agents, radioisotopes, radiopharmaceuticals, contrast agents, nanoparticles; enzymes, prosthetic groups, fluorescent materials, luminescent materials, and bioluminescent materials, such as, but not limited to, those described in U.S. patent No. 8,815,235, which is incorporated herein by reference in its entirety.

135. The therapeutic compound according to any one of embodiments 1-101, wherein the therapeutic compound does not comprise a radionuclide, such as but not limited to those described in U.S. patent No. 6,232,287, which is incorporated herein by reference in its entirety.

136. The therapeutic compound of any one of embodiments 1-101 that is not internalized by the donor cell to which it is bound.

137. The therapeutic compound of any one of embodiments 1-101, wherein the therapeutic compound does not enter the cell targeted by the specific targeting moiety.

138. The therapeutic compound of any one of embodiments 1-101, wherein the therapeutic compound does not kill the cells targeted by the specific targeting moiety.

139. The therapeutic compound according to any one of embodiments 1-101, wherein the therapeutic compound does not enter the cell to which the effector binding/modulating moiety binds.

140. The therapeutic compound of any one of embodiments 1-101 wherein the therapeutic compound does not kill the cells to which the effector binding/modulating moiety binds.

141. The therapeutic compound of any one of embodiments 1-101, wherein the therapeutic compound does not comprise an autoantigen peptide or polypeptide.

142. The therapeutic compound of any one of embodiments 1-101, wherein the therapeutic compound does not comprise an autoantigen peptide or polypeptide, e.g., does not comprise a peptide or polypeptide to which an autoantibody of the subject is directed.

143. The therapeutic compound according to any one of embodiments 1-101, wherein the therapeutic compound does not comprise an antibody molecule derived from a mammal (e.g., a human) having an autoimmune disorder.

144. The therapeutic compound according to any one of embodiments 1 to 101, wherein the therapeutic compound does not comprise an antibody molecule derived from a mammal (e.g. a human) having acute mucocutaneous PV.

145. The therapeutic compound of any one of embodiments 1-101, wherein the therapeutic compound does not comprise an antibody molecule derived from a mammal (e.g., a human) having goodpasture's disease.

146. The therapeutic compound of any one of embodiments 1-101, wherein the therapeutic compound does not comprise an antibody molecule derived from a mammal (e.g., a human) having pemphigus vulgaris.

141. The therapeutic compound of any one of embodiments 1-146, comprising a donor-specific targeting moiety.

142. The therapeutic compound of any one of embodiments 141, which is preferentially located at an implanted donor tissue relative to a recipient's tissue.

143. The therapeutic compound of embodiments 141-142, wherein the donor-specific targeting moiety provides a site-specific immune-privileged for a transplanted tissue (e.g., organ) from a donor.

144. The therapeutic compound of embodiments 141-143, wherein the donor-specific targeting moiety binds to a product (e.g., a polypeptide product) of an allele present at a locus in the donor that is not present at the locus in the recipient.

145. The therapeutic compound of any one of embodiments 141-144, wherein the donor-specific targeting moiety preferentially binds to an allele of a gene expressed on a donor tissue (e.g., a transplanted tissue, e.g., an organ) as compared to an allele of a gene expressed on a tissue of the subject.

146. The therapeutic compound of any one of embodiments 141-145, wherein the binding affinity of the donor-specific targeting moiety to an allele of a gene expressed on a donor tissue (e.g., a transplanted tissue, e.g., an organ) is at least 2, 4, 5, 10, 50, 100, 500, 1,000, 5,000, or 10,000 times greater than its affinity to an allele of a gene expressed on a tissue of a subject.

147. The therapeutic compound of any one of embodiments 141-146, wherein the donor-specific targeting moiety binds to a product (e.g., a polypeptide product) of an allele present at a locus in the donor that is not present at the locus in the recipient.

148. The therapeutic compound of any one of embodiments 141-147, wherein binding is sufficiently specific such that, e.g., at a clinically effective dose of the therapeutic compound, undesired, significant, or clinically unacceptable systemic immunosuppression occurs.

149. The therapeutic compound of any one of embodiments 141-148, wherein the therapeutic compound accumulates at the target site, e.g., binds to a donor-specific targeting moiety, thereby causing the therapeutic compound to accumulate at the target site.

150. The therapeutic compound of any one of embodiments 141-149, wherein the donor-specific targeting moiety binds to the product of an allele selected from a locus of table 2, e.g., an HLA locus, e.g., HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ or HLA-DR locus, which is present in the donor but not present in the recipient. HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ or HLA-DR locus.

151. The therapeutic compound of any one of embodiments 141-150, wherein donor-specific targeting moiety binds to an allele of HLAA, an allele of HLA-B, an allele of HLA-C, an allele of HLA-DP, an allele of HLA-, or an allele of HLA-.

152. The therapeutic compound of any one of embodiments 141-151, wherein the therapeutic compound is suitable for treating a subject having, about to have, or in need of a transplant.

153. The therapeutic compound of embodiment 152, wherein the transplant comprises all or a portion of an organ, such as a liver, kidney, heart, pancreas, thymus, skin, or lung.

154. The therapeutic compound of any one of embodiments 141-153, wherein the donor-specific targeting moiety comprises an antibody molecule.

155. The therapeutic compound of any one of embodiments 141-153, wherein donor-specific targeting moiety comprises a target-specific binding polypeptide, or a target ligand binding molecule.

156. The therapeutic compound of any one of embodiments 1-155 comprising a tissue-specific targeting moiety.

157. The therapeutic compound of embodiment 156, wherein the tissue-specific targeting moiety is a molecule that specifically binds to MAdCAM.

158. The therapeutic compound of embodiment 156 wherein the tissue-specific targeting moiety is an antibody that specifically binds MAdCAM.

159. The therapeutic compound according to any one of embodiments 156-158, wherein the therapeutic compound is suitable for treating a subject having, at risk of having, or at increased risk of having an autoimmune disorder (e.g., an autoimmune disorder as described herein).

160. The therapeutic compound according to any one of embodiments 156-159, wherein the therapeutic compound accumulates at the target site, e.g., binding to a tissue-specific targeting moiety results in the therapeutic compound accumulating at the target site.

161. The therapeutic compound of any one of embodiments 156-160, wherein the therapeutic compound is preferentially localized to a target tissue relative to other tissues of the subject.

162. The therapeutic compound of any one of embodiments 156-161, wherein the therapeutic compound provides site-specific immune-privileged protection for a target tissue in a subject, e.g., a target tissue that is experiencing, or is at risk of, an undesired immune attack (e.g., an immune disorder), or is at an elevated risk of an undesired immune attack.

163. The therapeutic compound of any one of embodiments 156-161, wherein the tissue-specific targeting moiety as a component of the therapeutic compound preferentially binds to a target tissue of a subject undergoing an undesired immune attack (e.g., an autoimmune disorder).

164. The therapeutic compound of any one of embodiments 156-163, wherein the tissue-specific targeting moiety binds a product, e.g., a polypeptide, that is not present outside the target tissue, or is present at a sufficiently low level such that at a therapeutic concentration of the therapeutic molecule, an unacceptable level of immunosuppression is not present or is substantially absent.

165. The therapeutic compound of any one of embodiments 156-164 wherein the tissue-specific targeting moiety binds to a product or a site on a product that is more abundant in a target tissue than in a non-target tissue.

166. The therapeutic compound of any one of embodiments 156-165 wherein the therapeutic compound binds to a product or site on a product that is substantially only present or expressed on a target tissue.

167. The therapeutic compound of any one of embodiments 156-166, wherein the product or site on the product to which the specific targeting moiety binds is sufficiently limited to the target tissue such that the subject does not suffer from unacceptable levels of systemic immunosuppression, e.g., clinically significant levels of systemic immunosuppression, at therapeutically effective levels of the therapeutic compound.

168. The therapeutic compound of any of embodiments 156-167, wherein the therapeutic compound preferentially binds to, e.g., has a binding affinity for, a target tissue or antigen that is greater than, e.g., at least 2, 4, 5, 10, 50, 100, 500, 1,000, 5,000, or 10,000 times greater than, the binding affinity of the therapeutic compound for a non-target tissue or antigen present outside of the target tissue.

169. The therapeutic compound of any of embodiments 156-168, wherein the tissue-specific targeting moiety binds to a product, e.g., a polypeptide product or a site on a product, present at a preselected site (e.g., a site of an undesired immune response in an autoimmune disorder).

170. The therapeutic compound of any one of embodiments 156-169, wherein the therapeutic compound is suitable for treating a patient having, or at risk of having, or at increased risk of having, type 1 diabetes.

171. The therapeutic compound of any one of embodiments 156-170, wherein the target tissue comprises pancreatic tissue (e.g., pancreatic islets or pancreatic beta cells), intestinal tissue (e.g., intestinal epithelial cells), renal tissue (e.g., renal epithelial cells), or hepatic tissue (e.g., hepatic epithelial cells).

172. A therapeutic compound according to any one of embodiments 156 to 171, wherein the effector binding/modulating or targeting moiety binds to a polypeptide selected from those described herein, such as those listed in table 3, e.g. SEZ6L2, LRP11, DISP2, SLC30a8, FXYD2, TSPAN7 or TMEM 27.

173. The therapeutic compound of any one of embodiments 156-168, wherein the therapeutic compound is suitable for treating a patient having, or at risk of having, or at increased risk of having, multiple sclerosis.

174. The therapeutic compound of embodiment 173, wherein the target tissue comprises CNS tissue, myelin sheath, or oligodendrocytes of myelin sheath.

175. The therapeutic compound according to any one of embodiments 173-174, wherein the effector binding/modulating moiety or targeting moiety binds a polypeptide selected from those described herein and including but not limited to table 3, e.g., MOG, PLP or MBP.

176. The therapeutic compound of any one of embodiments 156-168, wherein the therapeutic compound is suitable for treating a patient having, or at risk of or at increased risk of having myocarditis.

177. The therapeutic compound of embodiment 176, wherein the target tissue comprises cardiomyocytes, monocytes, macrophages or bone marrow cells.

178. The therapeutic compound according to embodiments 176 to 177 wherein the effector binding/modulating moiety binds or targets a moiety such as a polypeptide as described herein including but not limited to those selected from table 3, for example SIRPA (CD172 a).

179. The therapeutic compound according to any one of embodiments 156-168, wherein the therapeutic compound is suitable for treating a subject having, at risk of having, or at increased risk of having: inflammatory bowel disease, autoimmune hepatitis (AIH); primary Sclerosing Cholangitis (PSC); primary biliary cirrhosis; (PBC); or a transplant.

180. The therapeutic compound of any one of embodiments 156-168, wherein the subject has, is at risk of, or is at increased risk of having crohn's disease or ulcerative colitis.

181. The therapeutic compound of embodiment 179 or 180, wherein the target tissue comprises intestinal cells, such as intestinal epithelial cells; or hepatocytes, such as hepatic epithelial cells.

182. The therapeutic compound according to embodiments 179 to 181, wherein the effector binding/modulating moiety binds to a polypeptide as described herein, including but not limited to those selected from table 3, such as PD-1.

182. The therapeutic compound according to embodiments 179 to 181, wherein the effector binding/modulating moiety binds to a polypeptide as described herein, including but not limited to MAdCAM.

183. The therapeutic compound of any one of embodiments 156-168, wherein the therapeutic compound is suitable for treating a patient having, or at risk of having, or at increased risk of having rheumatoid arthritis.

184. The therapeutic compound of embodiment 183, wherein the target tissue comprises cardiomyocytes, monocytes, macrophages or bone marrow cells.

185. The therapeutic compound of embodiment 183 or 184, wherein the effector binding/modulating or targeting moiety binds to a polypeptide selected from table 3, e.g. SIRPA (CD172 a).

186. The therapeutic compound of any one of embodiments 156-185, wherein the tissue-specific targeting moiety comprises an antibody molecule.

187. The therapeutic compound of any one of embodiments 156-185, wherein the tissue-specific targeting moiety comprises a target-specific binding polypeptide, or a target ligand binding molecule.

188. The therapeutic compound of any one of embodiments 156-185, wherein the tissue-specific targeting moiety comprises a target-specific binding polypeptide that binds to MAdCAM.

189. The therapeutic compound of any one of embodiments 1-188, wherein the therapeutic compound binds to a cell surface molecule of an immune effector cell (e.g., a T cell, B cell, NK cell, or other immune cell) that diffuses the pro-immune response.

190. The therapeutic compound of any one of embodiments 1-189, wherein the therapeutic compound reduces the ability of immune effector cells (e.g., T cells, B cells, NK cells, or other immune cells) to diffuse pro-immune responses.

191. The therapeutic compound according to any one of embodiments 1-190, wherein the specific targeting moiety targets a mammalian target (e.g., a mammalian polypeptide) and the effector binding/modulating moiety binds/modulates a mammalian immune component, e.g., a human immune cell, e.g., a mammalian B cell, T cell, or macrophage.

192. The therapeutic compound according to any one of embodiments 1-192, wherein the specific targeting moiety targets a human target (e.g., a human polypeptide) and the effector binding/modulating moiety binds/modulates a human immune component, e.g., a human immune cell, e.g., a human B cell, T cell, or macrophage.

193. The therapeutic compound of any one of embodiments 1-193, wherein therapeutic compound is configured for use in a human.

194. The therapeutic compound according to any one of embodiments 1-191, wherein the therapeutic compound is configured for use in a non-human mammal.

195. The therapeutic compound of any one of embodiments 1-194, wherein the therapeutic compound (effector binding/modulating moiety) comprises a PD-1 agonist.

196. A method of treating a subject having an inflammatory bowel disease, the method comprising administering to a subject a therapeutic compound according to any one of embodiments 1 to 195 to treat the inflammatory bowel disease.

197. The method of embodiment 196, wherein the subject having inflammatory bowel disease has crohn's disease.

198. The method of embodiment 196, wherein the subject having inflammatory bowel disease has ulcerative colitis.

199. A method of treating a subject having autoimmune hepatitis, the method comprising administering to a subject a therapeutic compound according to any one of embodiments 1 to 195 to treat the autoimmune hepatitis.

200. A method of treating primary sclerosing cholangitis, the method comprising administering to a subject a therapeutic compound according to any one of embodiments 1 to 195 to treat the primary sclerosing cholangitis.

201. A method of treating type 1 diabetes, comprising administering a therapeutic compound according to any one of embodiments 1 to 195, thereby treating the subject to treat type 1 diabetes.

202. A method of treating a transplant subject comprising administering to the subject a therapeutically effective amount of the therapeutic compound according to any one of embodiments 1 to 195, thereby treating the transplant (recipient) subject.

203. A method of treating GVHD in a subject having transplanted donor tissue, the method comprising administering to the subject a therapeutically effective amount of a therapeutic compound according to any one of embodiments 1 to 195.

204. The method of embodiment 203, wherein prior to receiving the transplant; before symptoms of GVHD develop; after or concurrently with receiving the transplant; or administering a therapeutic compound to the subject after or concurrently with the onset of symptoms of GVHD.

205. A method of treating a subject having, at risk of having, or at increased risk of having an autoimmune disorder, the method comprising administering a therapeutically effective amount of a therapeutic compound according to any one of embodiments 1 to 195, thereby treating the subject.

206. The method of embodiment 205, wherein the subject has received, will receive, or is in need of allograft donor tissue.

207. The method of any one of embodiments 205-206, wherein the donor tissue comprises a solid organ, such as a liver, kidney, heart, pancreas, thymus, or lung.

208. The method of any one of embodiments 205-206, wherein the donor tissue comprises all or part of an organ, such as a liver, kidney, heart, pancreas, thymus, or lung.

209. The method of any one of embodiments 205-206, wherein the donor tissue comprises skin.

210. The method according to any one of embodiments 205-206, wherein the donor tissue does not comprise skin.

211. The method according to any one of embodiments 205-210, wherein the donor tissue presents or expresses the product of an allele of a locus that is not present or expressed in the subject.

212. The method of any one of embodiments 205 to 210, wherein the donor tissue presents or expresses a product of an allele selected from the loci of table 2, e.g., an HLA locus, e.g., HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ or HLA-DR locus, which allele is not present or expressed in the subject.

213. The method according to any one of embodiments 205 to 212, comprising introducing a transplanted tissue into a subject.

214. The method according to any one of embodiments 196-213, comprising monitoring the subject for immune cell inactivation (e.g., monitoring for undesired agonism of an immunosuppressive checkpoint molecule) at a site distant from the target site (e.g., in the peripheral circulation or lymphatic system).

215. The method according to any one of embodiments 196 to 214, comprising monitoring immune cell activation (e.g., monitoring undesired antagonism of an immunosuppressive checkpoint molecule) in the subject at a site distant from the target site (e.g., in the peripheral circulation or lymphatic system).

216. The method according to any one of embodiments 196-215, wherein in response to the monitoring result, the course of treatment of the subject is selected, e.g., increasing the dose of the therapeutic compound, decreasing the dose of the therapeutic compound, continuing treatment with the therapeutic compound without changing the dose.

217. The method of any one of embodiments 196-216, comprising administering to a recipient a compound according to embodiments 1-195.

218. The method according to any one of embodiments 196-216, wherein administering comprises systemic administration, e.g., to the peripheral circulatory system.

219. The method of any one of embodiments 196-216, wherein administering comprises local administration, e.g., to a target tissue, a donor tissue, or a site at which a target tissue or a donor tissue is or will be located.

220. The method of any one of embodiments 219, comprising administering a therapeutic compound to a recipient prior to introducing donor tissue to the recipient.

221. The method of any one of embodiments 219, comprising administering a therapeutic compound to a recipient after introducing donor tissue to the recipient.

222. The method of any one of embodiments 213, comprising administering the therapeutic compound to the recipient simultaneously with the introduction of the donor tissue to the recipient.

223. The method of embodiment 213, comprising contacting the therapeutic compound with the donor tissue prior to introducing the donor tissue into the recipient.

224. The method of any one of embodiments 213, comprising providing the therapeutic compound to the subject, wherein the transplanted tissue has been contacted with the therapeutic compound prior to introduction into the subject.

225. The method according to any one of embodiments 213, comprising contacting the therapeutic compound with the donor tissue after introducing the donor tissue into the recipient, e.g., by topical administration to the donor tissue.

226. The method according to any one of embodiments 196 to 226, comprising administering a therapeutic compound as provided herein such that the therapeutic level is present for at least 1, 5, 10, 14 or 28 days, e.g., consecutive or non-consecutive days.

227. The method of any one of embodiments 196-226, wherein the subject does not receive a non-targeted immunosuppressant.

228. The method of any one of embodiments 196-226, wherein the subject does not receive a non-targeted immunosuppressant at least 1, 15, 30, 60, or 90 days prior to initial administration of the therapeutic compound.

229. The method of any one of embodiments 213, wherein the subject does not receive the non-targeted immunosuppressant for at least 1, 15, 30, 60, or 90 days prior to introduction of the transplanted tissue.

230. The method of any one of embodiments 196-229, wherein the subject does not receive the non-targeted immunosuppressant for at least 1, 15, 30, 60, 90, or 180 days prior to initial administration of the therapeutic compound.

231. The method of any one of embodiments 196-229, wherein the subject does not receive the non-targeted immunosuppressant for at least 1, 15, 30, 60, 90, or 180 days prior to introduction of the transplanted tissue.

232. The method of any one of embodiments 196-231, comprising administering a non-targeted immunosuppressant to a subject.

233. The method of any one of embodiments 196-232, wherein the subject receives the non-targeted immunosuppressant at least 1, 15, 30, 60, or 90 days prior to initial administration of the therapeutic compound.

234. The method of embodiment 213, wherein the subject receives the non-targeted immunosuppressant at least 1, 15, 30, 60 or 90 days prior to introduction of the transplanted tissue.

235. The method of embodiment 234, wherein the subject receives the non-targeted immunosuppressant at least 1, 15, 30, 60, 90 or 180 days after initial administration of the therapeutic compound.

236. The method of any one of embodiments 196-235, wherein the subject receives the non-targeted immunosuppressant at least 1, 15, 30, 60, 90, or 180 days after introduction of the transplanted tissue.

237. The method according to any one of embodiments 196-235, wherein the subject receives the non-targeted immunosuppressant prior to but not more than 1, 15, 30, 60, 90 or 180 days prior to initial administration of the therapeutic compound.

238. The method of embodiment 213, wherein the subject receives the non-targeted immunosuppressant prior to introduction of the transplanted tissue but no more than 1, 15, 30, 60, 90, or 180 days.

239. The method of any one of embodiments 196-238, wherein the subject receives the non-targeted immunosuppressant after but not more than 1, 15, 30, 60, 90 or 180 days after initial administration of the therapeutic compound.

240. The method of embodiment 213, wherein the subject receives the non-targeted immunosuppressant after but not more than 1, 15, 30, 60, 90 or 180 days after introduction of the transplanted tissue.

241. The method of embodiment 213, wherein the subject is monitored for rejection of transplanted tissue.

242. The method of any one of embodiments 196-242, selecting a dose of the non-targeted immunosuppressant, or wherein in response to monitoring, selecting a dose of the non-targeted immunosuppressant.

243. The method of embodiment 242, wherein a dose is administered.

244. The method of embodiment 243, wherein the selected dose is zero, i.e., no non-targeted immunosuppressant is administered.

245. The method of embodiment 243, wherein the selected dose is not zero, i.e., no non-targeted immunosuppressant is administered.

246. The method of embodiment 243, wherein the dose is less than the dose administered without administration of the therapeutic compound.

247. The method according to any one of embodiments 196 to 246, wherein the subject is a mammal, e.g., a non-human mammal.

248. The method according to any one of embodiments 196 to 246, wherein the subject is a human.

249. The method of embodiment 213, wherein the donor and subject are mismatched at an HLA locus (e.g., a major or minor locus).

250. The method according to embodiment 249, wherein the subject is a mammal, e.g., a non-human mammal.

251. The method of embodiment 249, wherein the subject is a human.

252. A method of treating a subject having, at risk of having, or at increased risk of having an autoimmune disorder, the method comprising administering a therapeutically effective amount of a therapeutic compound according to any one of embodiments 1 to 195, thereby treating the subject.

253. The method of embodiment 252, wherein the providing of the therapeutic compound is initiated prior to the onset of symptoms of the autoimmune disorder or prior to the onset being identified.

254. The method according to any one of embodiments 252 to 253, wherein the provision of the therapeutic compound is initiated after the onset of symptoms of the autoimmune disorder or after the onset is identified.

255. The method according to any one of embodiments 252 to 254, wherein the autoimmune disorder comprises type 1 diabetes.

256. A therapeutic compound according to any one of embodiments 252-255, wherein the target tissue comprises pancreatic islet or pancreatic beta cells, intestinal tissue (e.g., intestinal epithelial cells), renal tissue (e.g., renal epithelial cells), or hepatic tissue (e.g., hepatic epithelial cells).

257. A therapeutic compound according to any one of embodiments 252 to 256, wherein an effector binding/modulating or targeting moiety binds to a polypeptide selected from table 3, e.g. a MAdCAM, SEZ6L2, LRP11, DISP2, SLC30a8, FXYD2, TSPAN7 or TMEM27 polypeptide.

258. The method according to any one of embodiments 252 to 257, wherein the providing of the therapeutic compound is initiated prior to the onset of symptoms of type 1 diabetes or prior to the onset being identified.

259. The method according to any one of embodiments 252-258, wherein providing the therapeutic compound is initiated prior to the subject having the preselected characteristic or symptom or prior to identifying the subject as having the preselected characteristic or symptom.

260. The method according to any one of embodiments 252-259, wherein the providing of the therapeutic compound begins after onset of symptoms of type 1 diabetes or after onset is identified.

261. The method according to any one of embodiments 252-260, wherein providing the therapeutic compound is initiated after the subject has a preselected characteristic or symptom or after the subject is identified as having a preselected characteristic or symptom.

262. The method according to any one of embodiments 252 to 261, wherein the therapeutic compound is a therapeutic compound according to any one of embodiments 1 to 195.

263 the method according to any one of embodiments 252 to 257, wherein the therapeutic compound is suitable for treating a patient suffering from, or at risk of suffering from or at increased risk of suffering from multiple sclerosis.

264. The method of embodiment 263, wherein the target tissue comprises CNS tissue, myelin sheath, or oligodendrocytes of myelin sheath.

265. The method according to any one of embodiments 263 or 264, wherein the effector binding/modulating or targeting moiety binds to a polypeptide selected from table 3, e.g. a MOG, PLP or MBP polypeptide.

266. The method according to any one of embodiments 263-265, wherein the providing of the therapeutic compound is initiated prior to the onset of a symptom of multiple sclerosis or prior to the onset being identified.

267. The method of any one of embodiments 263-265, wherein providing the therapeutic compound is initiated prior to the subject pre-selecting the characteristic or symptom or prior to identifying the subject pre-selected characteristic or symptom.

268. The method according to any one of embodiments 263-265, wherein the providing of the therapeutic compound is initiated after the onset of a symptom of multiple sclerosis or after the onset is identified.

269. The method of any one of embodiments 263-265, wherein providing the therapeutic compound is initiated after the subject has a preselected characteristic or symptom or after identifying the subject as having a preselected characteristic or symptom.

270. The method according to any one of embodiments 263 to 269, wherein the therapeutic compound is a therapeutic compound according to any one of embodiments 1 to 195.

271. The method according to any one of embodiments 252 to 257, wherein the therapeutic compound is suitable for treating a patient suffering from, or at risk of suffering from, or at increased risk of suffering from myocarditis.

272. The method of embodiment 271, wherein the target tissue comprises cardiomyocytes, monocytes, macrophages or bone marrow cells.

273. The method according to embodiment 271 or 272, wherein the effector binding/modulating or targeting moiety binds to a polypeptide selected from table 3, for example a SIRPA (CD172a) polypeptide.

274. The method according to any one of embodiments 271 to 273, wherein the providing of the therapeutic compound is initiated prior to the onset of symptoms of myocarditis or prior to identifying the onset.

275. The method according to any one of embodiments 271-273, wherein providing the therapeutic compound is initiated prior to the subject having the preselected characteristic or symptom or prior to identifying the subject as having the preselected characteristic or symptom.

276. The method according to any one of embodiments 271 to 273, wherein the providing of the therapeutic compound begins after the onset of symptoms of myocarditis or after the onset is identified.

277. The method according to any one of embodiments 271-273, wherein providing the therapeutic compound is initiated after the subject has a preselected characteristic or symptom or after the subject is identified as having a preselected characteristic or symptom.

278. The method according to any one of embodiments 271 to 277, wherein the therapeutic compound is a therapeutic compound according to any one of embodiments 1 to 195.

279. The method according to any one of embodiments 252 to 257, wherein the therapeutic compound is suitable for use in the treatment of a patient suffering from, or at risk of suffering from or at increased risk of suffering from rheumatoid arthritis.

280. The method of embodiment 279, wherein the target tissue comprises cardiomyocytes, monocytes, macrophages or bone marrow cells.

281. The method according to embodiment 279 or 280, wherein the effector binding/modulating or targeting moiety binds to a polypeptide selected from table 3, for example a SIRPA (CD172a) polypeptide.

282. The method according to embodiments 279 to 281, wherein the provision of the therapeutic compound is initiated prior to the onset of symptoms of multiple sclerosis or prior to the identification of the onset.

283. The method of embodiments 279-281, wherein providing the therapeutic compound is initiated before the subject has a preselected characteristic or symptom or before the subject is identified as having a preselected characteristic or symptom.

284. The method according to embodiments 279 to 281, wherein the provision of the therapeutic compound is initiated after the onset of symptoms of rheumatoid arthritis or after the onset is identified.

285. The method of embodiments 279-281, wherein providing the therapeutic compound is initiated after the subject has a preselected characteristic or symptom or after the subject is identified as having a preselected characteristic or symptom.

286. The method according to embodiments 279 to 285, wherein the therapeutic compound is a therapeutic compound according to any one of embodiments 1 to 195.

287. The method according to any one of embodiments 196-286, comprising monitoring a subject for immune cell inactivation (e.g., monitoring for undesired agonism of an immunosuppressive checkpoint molecule) at a site distant from a target site (e.g., in the peripheral circulation or lymphatic system).

288. The method according to any one of embodiments 196 to 287, comprising monitoring immune cell activation (e.g., monitoring undesired antagonism of an immunosuppressive checkpoint molecule) in the subject at a site distant from the target site (e.g., in the peripheral circulation or lymphatic system).

289. The method according to any one of embodiments 196-288, wherein the course of treatment of the subject is selected in response to the monitoring result, e.g., increasing the dose of the therapeutic compound, decreasing the dose of the therapeutic compound, continuing treatment with the therapeutic compound without changing the dose.

290. The method of any one of embodiments 196-289, wherein the target tissue of the subject is monitored for autoimmune attack.

291. The method of embodiment 290, wherein the dose of the therapeutic compound is selected in response to monitoring.

292. The method of embodiment 291, wherein a dose is administered.

293. The method of embodiment 290, wherein the selected dose is zero, i.e., administration of the therapeutic compound is discontinued.

294. The method of embodiment 290, wherein the selected dose is not zero.

295. The method of embodiment 290, wherein the selected dose is an escalated dose.

296. The method of embodiment 290, wherein the selected dose is a reduced dose.

297. The method of any one of embodiments 196-296, wherein administering comprises systemic administration, e.g., administration to the peripheral circulatory system.

298. The method of any one of embodiments 196-297, wherein administering comprises topical administration, e.g., to a target tissue.

299. The method according to any one of embodiments 196-298 comprising administering a therapeutic compound provided herein such that therapeutic levels are present for at least 1, 5, 10, 14, or 28 days, e.g., consecutive or non-consecutive days.

300. The method according to any one of embodiments 196 to 299, wherein the subject is a mammal, e.g. a non-human mammal.

301. The method according to any one of embodiments 196 to 299, wherein the subject is a human.

302. A nucleic acid molecule or a plurality of nucleic acid molecules encoding a therapeutic compound according to any one of embodiments 1 to 195.

303. A vector or vectors comprising a nucleic acid molecule according to embodiment 302.

304. A cell comprising the nucleic acid molecule of embodiment 302 or the vector of embodiment 303.

305. A method of making a therapeutic compound comprising culturing a cell according to embodiment 304 to make a therapeutic compound.

306. A method of making a nucleic acid sequence encoding a therapeutic compound according to any one of embodiments 1-195, comprising:

a) providing a vector comprising a sequence encoding a targeting moiety and inserting into the vector sequence encoding an effector binding/modulating moiety to form a sequence encoding a therapeutic compound; or

b) Providing a vector comprising a sequence encoding an effector binding/modulating moiety and inserting into the vector sequence encoding a targeting moiety to form a sequence encoding a therapeutic compound,

thereby preparing a sequence encoding a therapeutic compound.

307. The method of embodiment 306, wherein the targeting moiety is selected according to the needs of the subject.

308. The method according to embodiment 306 or 307, wherein the effector binding/modulating moiety is selected according to the need of the subject.

309. The method according to any one of embodiments 306 or 307, further comprising expressing a sequence encoding a therapeutic compound to produce a therapeutic compound.

310. The method according to any one of embodiments 306-309, further comprising transferring the sequence or polypeptide prepared from the sequence to another entity, e.g., a healthcare provider that administers a therapeutic compound to a subject.

311. A method of treating a subject, comprising:

obtaining (e.g., receiving from another entity) a therapeutic compound or a nucleic acid encoding a therapeutic compound prepared by any one of the methods provided herein, but not limited to, embodiments 306-310;

administering to the subject a therapeutic compound or a nucleic acid encoding a therapeutic compound,

thereby treating the subject.

312. The method of embodiment 311, further comprising identifying the therapeutic compound or the nucleic acid encoding the therapeutic compound to another entity, e.g., an entity that will make the therapeutic compound, or the nucleic acid encoding the therapeutic compound.

313. The method of embodiment 311 or 312, further comprising claiming the therapeutic compound or the nucleic acid encoding the therapeutic compound from another entity (e.g., the entity that prepared the therapeutic compound or the nucleic acid encoding the therapeutic compound).

314. The method according to any one of embodiments 311 to 333, wherein the subject has an autoimmune disorder and the therapeutic compound does not include an autoantigenic peptide or polypeptide of the autoimmune disorder, e.g., does not include a peptide or polypeptide to which an autoantibody of the subject is directed.

The following examples illustrate the compounds, compositions and methods described herein, but are not limiting. Other suitable modifications and adaptations known to those skilled in the art are within the scope of the following embodiments.

Examples

Example 1 HLA-targeted PD-1 agonist therapeutic compounds.

Engineered HLA-targeted PD-1 agonist therapeutics

Binding knots specific for HLA-A2 were obtained by cloning the variable regions of Ig heavy and light chains from BB7.2 hybridoma (ATCC) and converting into single chain Ab (scFv)And (4) domain formation. The activity and specificity of the scFv can be confirmed by assessing binding of BB7.2 to cells expressing HLA-A2 compared to cells expressing other HLA-A alleles. The minimum PD-L1 residues required for PD-1 binding activity were identified by systematically evaluating the need for amino acids 3 'and 5' of the PD-L1 IgV domain corresponding to amino acids 68-114. Expression constructs were designed and proteins were synthesized and purified and tested for PD-1 binding activity by Biacore. The minimum essential amino acid required for the PD-L1 IgV domain to bind PD-1 is called PD-L1-IgV. To generate the BB7.2 scFv and PD-L1-IgV bispecific molecule, VL was synthetically encoded with a domain arrangement_BB7.2-VH_BB7.2DNA fragment of bispecific single chain antibody BB7.2x PD-L1-IgV of-PD-L1-IgV-4 Fc and cloned into an expression vector containing the DHFR selection cassette.

Expression vector plasmid DNA was transiently transfected into 293T cells and bb7.2x PD-L1-IgV bispecific antibody was purified from the supernatant using a protein a/G column. Bb7.2x PD-L1-IgV bispecific antibody integrity was assessed by polyacrylamide gel. Binding of the BB7.2 scFv domain to HLA-A2 and binding of the PD-L1-IgV domain to PD-1 was assessed by ELISA and cell-based FACS assays.

The Mixed Lymphocyte Reaction (MLR) assay was used to assess the in vitro function of bb7.2x PD-L1-IgV bispecific antibodies. In the 96-well plate format, each well aliquot is from HLA-A2⁺100,000 irradiated human PBMCs from donors were used as activators. Then HLA-A1 is added^-Responder T cells were added with increasing amounts of bb7.2x PD-L1-IgV bispecific antibody. The ability of responder T cells to proliferate within 72 hours was assessed by BrdU incorporation, and IFNg and IL2 cytokine production were additionally assessed in the co-culture supernatants as assessed by ELISA. BB7.2x PD-L1-IgV bispecific antibodies were found to inhibit the MLR response, e.g., by inhibiting HLA-A2^-Response T cell proliferation and cytokine production.

The in vivo function of the bb7.2x PD-L1-IgV bispecific antibody was evaluated using a murine model of skin allograft tolerance. A C57BL/6-Tg (HLA-A2.1)1Enge/J (Jackson Laboratories, Bar Harbor Maine) mouse strain was crossed with Balb/cJ, in which the F1 progeny expressed the HLA-A2.1 transgene and served as an allograft donor. C57BL/6J mice were shaved and surgically implanted with skin removed from euthanized C57BL/6-Tg (HLA-A2.1)1Enge/Jx Balb/cJ F1 mice. At the same time, the host mice began receiving intraperitoneal injections of the bb7.2x PD-L1-IgV bispecific antibody engineered to contain only murine IgG1Fc or BB7.2 or only PD-L1-IgV control. Skin allograft rejection or acceptance was monitored over a period of 30 days, where the host was euthanized and the lymph node and lymphocyte population where the allograft resides quantified.

Example 2: as effector domains, CD39 and/or CD73 that produce purinergic halos around cell types or tissues of interest

A catalytically active fragment of CD39 and/or CD73 is fused to a targeting domain. After binding and accumulation at the target site, CD39 phosphorylates ATP to AMP. Upon binding and accumulation at the target site, CD73 dephosphorylates extracellular AMP to adenosine. Soluble catalytically active forms of CD39 suitable for use herein have been found to circulate in human and murine blood, see, e.g., Yegutkin et al faeb j.2012, month 9; 26(9):3875-83. Soluble recombinant CD39 fragments are also described in Inhibition of platelet function by recombinant soluble ecto-ADPase/CD39, Gayle et al, J Clin invest.1998, 5.1.d; 101(9):1851-1859. Suitable CD73 molecules include the soluble form of CD73, which can be shed from endothelial cell membranes by proteolytic cleavage or hydrolysis of GPI anchors using shear stress, see, for example, references: yegutkin G, Bodin P, Burnstock G.Effect of shear stress on the release of soluble ecto-enzymes ATPase and 5' -nucleotidase ion with endogenesis ATPf plasma vascular cells Br J Pharmacol 2000; 129:921-6.

Local catalysis of ATP to AMP or AMP to adenosine will deplete the local energy stores required for fulminant T effector cell function. Treg function should not be affected by ATP depletion because they rely on oxidative phosphorylation to achieve energy requirements (less ATP is required), whereas T memory and other effector cells should be affected because they rely on glycolysis (requiring high ATP usage) to perform fulminant functions.

Example 3: antibody-induced PD-1 signaling was measured.

Jurkat cells stably expressed 2 constructs: 1) a human PD-1 polypeptide fused to b-galactosidase, which may be referred to as an "enzyme donor" and 2) a SHP-2 polypeptide fused to b-galactosidase, which may be referred to as an "enzyme receptor". The PD-1 antibody is contacted with the cell, and when PD-1 is engaged, SHP-2 is recruited to PD-1. The enzyme acceptor and enzyme donor form a fully active b-galactosidase that can be measured. This assay can be used to demonstrate activation of PD-1 signaling.

Example 4: PD-1 agonism was measured. Without being bound by any particular theory, PD-1 agonists inhibit T cell activation, and PD-1 agonists inhibit anti-CD 3-induced T cell activation. Human or mouse cells were pre-activated with PHA (for human T cells) or ConA (for mouse T cells) so that they expressed PD-1. T cells can then be "reactivated" with anti-CD 3 in the presence of anti-PD-1 (or PD-L1) for use in PD-1 agonism assays. T cells receiving PD-1 agonist signaling in the presence of anti-CD 3 will show reduced activation relative to anti-CD 3 stimulation alone. Activation can be read by proliferation or cytokine production (IL-2, IFNg, IL-17) and possibly by other markers such as the CD69 activation marker.

Example 5 expression and function of anti-MAdCAM/mouse PD-L1 fusion protein was not affected by molecular configuration.

The bispecific fusion molecule comprising an anti-mouse MAdCAM Ab/mouse PD-L1 molecule was expressed in two orientations. The first orientation consisted of an anti-mouse MAdCAM IgG and mouse PD-L1 fused at the c-terminus of its heavy chain. The second orientation consisted of mouse PD-L1 fused at the N-terminus of the IgFc domain, and an anti-mouse MAdCAM scFv fused at the c-terminus. Both molecules were found to be well expressed in mammalian expression systems. It has also been found that molecules can bind to their respective binding partners MAdCAM or PD-1 in both directions simultaneously. These results indicate that a molecule consisting of an anti-MAdCAM antibody fused to PD-L1 can be expressed in the following configuration: wherein PD-L1 is fused to Fc at the N or C terminus and retains appropriate functional binding activity.

Briefly, a pTT5 vector containing a single gene encoding a single polypeptide with mouse PD-L1 fused to the N-terminus of the human IgG1Fc domain and anti-MAdCAM scFvMECA-89 fused to the c-terminus was transfected into HEK293Expi cells. Alternatively, both plasmids were co-transfected at equimolar ratios. The first plasmid encodes the light chain of MECA-89, and the second plasmid encodes the full-length IgG1 heavy chain of MECA-89 with a c-terminal fusion of mouse PD-L1. After 5-7 days, cell culture supernatants expressing the molecules were harvested and clarified by centrifugation and filtration through a 0.22um filtration device. The bispecific molecule is captured on the proA resin. The resin was washed with PBS pH 7.4 and the captured molecules were eluted using 100mM glycine pH 2.5, neutralized with one tenth volume of 1M Tris pH 8.5. The protein buffer was exchanged into PBS pH 7.4 and analyzed by size exclusion chromatography on Superdex 2003.2/300. 1ug of purified material was analyzed by reducing and non-reducing SDS-PAGE on Bis-Tris 4-12% gels.

Both proteins (regardless of orientation) were expressed at more than 10mg/L and were more than 95% monodisperse after purification as shown by size exclusion chromatography and reduced/non-reduced SDS-PAGE. Thus, this demonstrates the generation and activity of bifunctional bispecific molecules with different immunomodulators and tissue targeting moieties at the N-and C-termini of the Fc domain. This also shows in particular that PD-1 agonists and binding partners can be expressed at the N-terminus or C-terminus of an Ig Fc domain.

Example 6 bispecific molecules comprising a PD-1 agonist prototype tethered to MAdCAM can bind both MAdCAM and PD-1.

Briefly, the immunoadsorbent plates were coated with mouse PD-1(75 ul/well) at a concentration of 1ug/mL in PBS pH 7.4 and incubated overnight at 4 ℃. Each well was washed three times with PBS pH 7.4 containing 0.05% Tween-20 (wash buffer) and then blocked with 200 ul/well 1% BSA in PBS pH 7.4 (blocking buffer) for 2 hours at room temperature. After three washes with wash buffer, two bispecific molecules comprising the PD-1 agonist prototype at the N-or C-terminus were diluted to 1nM, 10nM and 100nM in PBS (assay buffer) containing 1% BSA and 0.05% Tween-20. The diluted material was added at 75 ul/well to the mouse PD-1 coated plate for 1 hour at room temperature. After three washes with wash buffer, mouse MAdCAM was added to the plate at 75 ul/well at a concentration of 10nM in assay buffer for 1 hour at room temperature. After three washes with wash buffer, goat biotinylated anti-mouse MAdCAM polyclonal antibody diluted to 0.5ug/mL in assay buffer was added to the plate at 75 ul/well for 1 hour at room temperature. After three washes with wash buffer, high sensitivity streptavidin HRP diluted 1:5000 in assay buffer was added to the plate at 75 ul/well for 15 minutes at room temperature. After washing three times with wash buffer and 1 time with wash buffer (without tween-20), the assay was developed with TMB and stopped with 1 NHCL. OD 450nm was measured. The experiment included appropriate controls for nonspecific binding to the plate/block in the absence of mouse PD-1, as well as a no MAdCAM control and a monospecific control, which failed to bridge between mouse PD-1 and mouse MAdCAM.

The results show that both bispecific molecules were able to interact simultaneously with mouse MAdCAM and mouse PD-L1 at concentrations of 1nM, 10nM and 100nM, whereas the monospecific controls did not produce a bridging signal. In addition, in the absence of mouse PD-1 on the plate surface, no compound bound to MAdCAM at any of the concentrations tested, indicating that no test compound is interacting non-specifically with the plate surface. Thus, these results demonstrate that bispecific molecules targeted to bind to MAdCAM and PD-1 can successfully bind to both molecules. Although experiments were performed with PD-L1 as a surrogate for the PD-1 antibody, it is expected that the PD-1 antibody will function in a similar manner.

Example 7 bispecific PD-L1 prototype molecules inhibited T cells in a PD-1 agonist assay.

Bispecific molecules that mimic PD-1 agonist antibodies were tested to demonstrate that PD-1 agonism can inhibit T cells. Briefly, 7 week old female C57LB/6 mice were sacrificed and their spleen cells were isolated. Splenocytes were exposed to ConA for 3 days and then to anti-CD 3 in the presence or absence of PD-1 type molecules, which in this example were PD-L1 bispecific molecules tethered to the plate using anti-human IgG. T cells were then introduced into the PD-L1 bispecific molecule. PD-L1, which mimics PD-1 antibody, was found to be a T cell agonist and to inhibit T cell activation. The same experiment was repeated using a PD-L1 bispecific molecule fused to an anti-MAdCAM antibody, which was tethered to the plate by interaction with the MAdCAM coated plate. The PD-1 agonist mimetic PD-L1/anti-MAdCAM antibody was found to be a potent agonist of T cell activity. These results indicate that bispecific molecules that mimic the PD-1 antibody/MAdCAM fusion protein can exert functional inhibitory signaling to primary mouse T cell blast cells when the molecule is captured by MAdCAM antibody components at the ends of the molecule.

Example 8: bispecific PD-1 prototype molecules with different tissue tethers could inhibit T cells in a PD-1 agonist assay. The fusion molecule of PD-L1 was used as a surrogate for PD-1 antibodies and linked to class I H-2Kk antibodies. MHC class I H-2Kk tethered PD-L1 molecules had functional binding similar to the data described in examples 6 and 7. Briefly, splenocytes from C57Bl/6 mice were stimulated with concanavalin A (ConA) and IL-2 for 3 days. Plates were coated with anti-CD 3(2C11) at 4C overnight and washed. Plates were coated with anti-human IgG at 37C for 3 hours and washed. Monospecific anti-H-2 Kk (16-3-22) or bispecific anti-H-2 Kk: mPD-L1 were added and incubated at 37C for 3 hours and washed. All test articles contained a human IgG1-Fc portion. PBS (no Tx) was added to determine assay background. ConA mother cells were washed 2 times, added to the plate and incubated at 37C. The supernatant was removed after 24 hours. IFNg content was determined by MSD. After 48 hours, Cell viability/metabolism was analyzed by Cell Titer-glo. MHC class I tethered PD-L1 bispecific, when captured by IgG Fc domain, can attenuate T cell activation in mouse PD-1 agonism assay. Thus, this example demonstrates that different bispecific proto-molecules can exert functional inhibitory signaling to primary mouse T cell blasts when the molecules are captured by different tissue tethers (in this case mouse antibodies to MHC class I H-2 Kk). Thus, this data indicates that the tether is not specific for MAdCAM and can be used with other molecules that can serve as targeting moieties as provided herein.

Example 9 PD-1 agonists can induce signaling in Jurkat cells

Jurkat cells expressing human PD-1 fused to the beta-galactosidase donor and SHP-2 fused to the beta-galactosidase receptor were added to the plate under test conditions and incubated for 2 hours. Agonist PD-1 antibodies induce signaling and SHP-2 recruitment, enzyme complementation and formation of active β -galactosidase enzyme. Beta-galactosidase substrate was added and chemiluminescence was measured on a standard light plate reader. Agonism is measured by chemiluminescence, where more chemiluminescence measured indicates greater agonism.

The agonism of the PD-1/MAdCAM bispecific molecule was measured in this assay. Cl10(UCB) and CC-90006(Celgene/Anaptys) were used as PD-1 agonist antibodies. Both are active and exhibit PD-1 agonism in a functional assay in the form of an Ig capture assay. Briefly, plates were coated with anti-human IgG at 4C and washed. Either anti-Tetanus Toxin (TT) or the benchmark agonist anti-PD-1 monoclonal antibody Cl.10 or CC-90006 was added and incubated at 37C for 1 hour and washed. All test articles contained human IgG 1-Fc. Media (no Tx) was added to determine assay background. The plate was washed 3 times. Jurkat cells expressing human PD-1 fused to the b-galactosidase donor and SHP-2 fused to the b-galactosidase receptor were added and incubated for 2 hours. Agonist PD-1 antibodies induce signaling and SHP-2 recruitment, enzyme complementation and formation of active b-galactosidase enzyme. B-galactosidase substrate was added and chemiluminescence was measured on a standard light plate reader. Two human PD-1 agonist antibodies (Cl10 and CC-90006) bind and induce signaling (an alternative to agonism) in a modified Jurkat reporter assay. Thus, the assay is a functional PD-1 agonism assay. Cl10 MECA89(MECA89 is a known MADCAM antibody) is a novel bispecific molecule produced by fusing the MADCAM antibody MECA89[ scFv ] to the C-terminus of the Cl10 heavy chain. The fusion protein was found to be active and when captured by the IgG Fc domain, showed PD-1 agonism in a functional assay, as was the Cl10 protein alone. However, only Cl10: MECA89 was active in the functional assay format using MAdCAM protein as capture (the monospecific component did not signal).

Briefly, plates were coated with anti-human IgG or recombinant mmadc cam-1 overnight at 4C and washed. Monospecific anti-Tetanus Toxin (TT), anti-MAdCAM-1 (MECA39) or agonist anti-PD-1 (Cl10) or bispecific Cl10: MECA89 were added and incubated at 37C for 1 hour and washed. All test articles contained a human IgG1-Fc portion. PBS (no Tx) was added to determine assay background. The plate was washed 2 times. Jurkat cells expressing human PD-1 fused to the b-galactosidase donor and SHP-2 fused to the b-galactosidase receptor were added and incubated for 2 hours. Agonist PD-1 antibodies induce signaling and SHP-2 recruitment, enzyme complementation and formation of active b-galactosidase enzyme. B-galactosidase substrate was added and chemiluminescence was measured on a standard light plate reader. As a result: both Cl10 and MAdCAM tethered Cl10 bispecific molecules can induce PD-1 signaling in the Jurkat reporter assay when the plate is coated with anti-IgG Fc capture, but only MAdCAM tethered bispecific molecules can induce PD-1 signaling in the reporter assay when the plate is coated with recombinant MAdCAM protein. These results indicate that molecules tethered with MAdCAM and containing PD-1 agonist antibodies are functional, similar to the results shown with PD-L1 as a PD-1 agonist surrogate.

Example 10: generation of PD-1 agonist antibodies

PD-1 deficient mice are immunized with mouse PD-1 under conditions that generate an immune response against PD-1. 54 hybridomas were generated and identified that bound mouse PD-1. Antibodies produced by the different hybridomas were analyzed for T cell agonism according to the methods described in examples 4 and 6. Of the 54 hybridomas, at least 6 were identified as PD-1 agonists. The binding of the antibodies on PD-1 was also tested and they were found to bind at the same site as the PD-L1 binding site.

Briefly, binding to the PD-L1 binding site was determined using the following assay. The immunoadsorption plates were coated overnight with 75. mu.L of 1 XPBS containing recombinant mouse PD-L1-Fc (2. mu.g/mL), pH 7.4. The plates were then washed 3 times with 1x PBS and blocked with 1x PBS supplemented with 1% BSA for 2 hours at room temperature. Recombinant mouse PD-1-Fc (1nM) was incubated with 100nM of the indicated anti-mouse PD-1 antibody in 1 XPBS (assay buffer) supplemented with 1% BSA and 0.05% Tween20 for 1 hour at room temperature with shaking. After blocking, plates were washed 3 times with 1x PBS supplemented with 0.05% Tween20 PBST and antibody-PD-1 conjugate incubated with plate-bound mouse PD-L1. After washing away unbound PD-1 with PBST, the plate was incubated with 75. mu.L of biotinylated polyclonal anti-PD-1 antibody (0.5. mu.g/mL) in assay buffer and then amplified with 1:5000 streptavidin HRP, also diluted in assay buffer. Plates were washed three times with PBST, then three times with 1 × PBS, then 100 μ L of LTMB was added, followed by 100 μ L of 1M HCl to stop the color development. The absorbance was read at 450nm and normalized to the binding of PD-1 to PD-L1 in the absence of antibody. The results show that the active antibody binds to the PD-L1 binding site. The inactivated antibody does not bind to the PD-L1 binding site. Thus, this example demonstrates the ability to produce anti-PD-1 antibodies as agonists in addition to the previously identified PD-1 agonist antibodies described herein.

Example 11: the tethered anti-PD-1 antibody acts as a PD-1 agonist.

Human antibody scFv phage libraries were panned against recombinant human, mouse and cynomolgus monkey PD-1 proteins in an iterative selection round to enrich for antibody clones that recognized all three of the above species orthologs of PD-1. The scFv clones were constructed in the nt-VH-linker-VL-ct format and fused to the M13 phage surface via pIII coat protein. After selection, the cloned scFv was screened for binding to human, mouse and cynomolgus PD-1 expressed on the cell surface of CHO cells. Clones found to cross-react with all three cell surface expressed PD-1 species orthologs were converted to a human IgG1 version using standard molecular biology techniques, where each molecule consists of a total of four polypeptide chains (2 heavy chains and 2 light chains). As provided, the two light chains are identical to each other, and the two heavy chains are identical to each other.

Two identical heavy chains homodimerize, and two identical light chains pair with each heavy chain to form a complete human IgG 1. The Fc domain contains L235A, L236A, and G237A mutations to eliminate FcyR interactions. The transformed human IgG1 anti-PD-1 antibody was transfected and expressed in HEK293Expi cells and purified by protein a chromatography. Protein concentration was determined using a nanodrop spectrophotometer coupled with an antibody specific extinction coefficient. Antibodies were formulated in PBS pH 7.4.

The anti-PD-1 antibodies were then tested for agonist activity in the Jurkat assay described herein. Briefly, the tissue culture plate is coated or uncoated with anti-IgG. For the captured format, test article or control was added at 100nM, 25nM or 12.5nM to anti-IgG coated wells and incubated at 37C for 3 hours. Plates were washed and Jurkat PD-1 cells were added. For the soluble format, soluble test articles or controls were added at 100nM, 25nM, or 12.5nM to wells already containing Jurkat PD1 cells. Luminescence was measured in a plate reader. The results show that nine of the twelve human/mouse cross-reactive PD-1 antibodies showed dose-dependent activity in the Jurkat assay when the anti-PD-1 antibody was captured by anti-IgG, but did not show dose-dependent activity in soluble form. This data indicates that anti-PD-1 antibodies can act as agonists when tethered to their target by a targeting moiety.

In summary, without being bound by any particular theory, the data provided herein indicate that PD-1 agonist/MAdCAM bispecific molecules can bind to MAdCAM and PD-1 and also agonize T cell activity. Thus, the molecules may be used to treat various disorders provided herein, and provide local and/or tissue-specific immune modulation and down-regulation of T cell responses.

The disclosures of each patent, patent application, and publication cited herein are hereby incorporated by reference in their entirety. Although various embodiments have been disclosed with reference to particular aspects, other aspects and variations of these embodiments may be devised by others skilled in the art without departing from the true spirit and scope of the invention. It is intended that the following claims be interpreted to embrace all such aspects and their equivalents.

Sequence listing

<110> Pandean Therapeutics, Inc.)

Joanni L Wayini (VINEY, Joane L.)

Nersen-Cikinson-Scott (HIGGINSON-SCOTT, Nathan)

Mijia-Bensen (BENSON, Micah)

Allen Kelin (CRANE, Alan)

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Ser Met Cys Ile Leu Asn Gly Gly Gly Ile Arg Ser Pro Ile Asp Glu

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Arg Asn Asn Gly Thr Ile Thr Trp Glu Asn Leu Ala Ala Val Leu Pro

405 410 415

Phe Gly Gly Thr Phe Asp Leu Val Gln Leu Lys Gly Ser Thr Leu Lys

420 425 430

Lys Ala Phe Glu His Ser Val His Arg Tyr Gly Gln Ser Thr Gly Glu

435 440 445

Phe Leu Gln Val Gly Gly Ile His Val Val Tyr Asp Leu Ser Arg Lys

450 455 460

Pro Gly Asp Arg Val Val Lys Leu Asp Val Leu Cys Thr Lys Cys Arg

465 470 475 480

Val Pro Ser Tyr Asp Pro Leu Lys Met Asp Glu Val Tyr Lys Val Ile

485 490 495

Leu Pro Asn Phe Leu Ala Asn Gly Gly Asp Gly Phe Gln Met Ile Lys

500 505 510

Asp Glu Leu Leu Arg His Asp Ser Gly Asp Gln Asp Ile Asn Val Val

515 520 525

Ser Thr Tyr Ile Ser Lys Met Lys Val Ile Tyr Pro Ala Val Glu Gly

530 535 540

Arg Ile Lys Phe Ser Thr Gly Ser His Cys His Gly Ser Phe Ser Leu

545 550 555 560

Ile Phe Leu Ser Leu Trp Ala Val Ile Phe Val Leu Tyr Gln

565 570

<210> 3

<211> 290

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 3

Met Arg Ile Phe Ala Val Phe Ile Phe Met Thr Tyr Trp His Leu Leu

1 5 10 15

Asn Ala Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr

20 25 30

Gly Ser Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu

35 40 45

Asp Leu Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile

50 55 60

Ile Gln Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser

65 70 75 80

Tyr Arg Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn

85 90 95

Ala Ala Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr

100 105 110

Arg Cys Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val

115 120 125

Lys Val Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val

130 135 140

Asp Pro Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr

145 150 155 160

Pro Lys Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser

165 170 175

Gly Lys Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn

180 185 190

Val Thr Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr

195 200 205

Cys Thr Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu

210 215 220

Val Ile Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Thr His

225 230 235 240

Leu Val Ile Leu Gly Ala Ile Leu Leu Cys Leu Gly Val Ala Leu Thr

245 250 255

Phe Ile Phe Arg Leu Arg Lys Gly Arg Met Met Asp Val Lys Lys Cys

260 265 270

Gly Ile Gln Asp Thr Asn Ser Lys Lys Gln Ser Asp Thr His Leu Glu

275 280 285

Glu Thr

290

<210> 4

<211> 313

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 4

Met Val Val Met Ala Pro Arg Thr Leu Phe Leu Leu Leu Ser Gly Ala

1 5 10 15

Leu Thr Leu Thr Glu Thr Trp Ala Gly Ser His Ser Met Arg Tyr Phe

20 25 30

Ser Ala Ala Val Ser Arg Pro Gly Arg Gly Glu Pro Arg Phe Ile Ala

35 40 45

Met Gly Tyr Val Asp Asp Thr Gln Phe Val Arg Phe Asp Ser Asp Ser

50 55 60

Ala Cys Pro Arg Met Glu Pro Arg Ala Pro Trp Val Glu Gln Glu Gly

65 70 75 80

Pro Glu Tyr Trp Glu Glu Glu Thr Arg Asn Thr Lys Ala His Ala Gln

85 90 95

Thr Asp Arg Met Asn Leu Gln Thr Leu Arg Gly Tyr Tyr Asn Gln Ser

100 105 110

Glu Ala Ser Ser His Thr Leu Gln Trp Met Ile Gly Cys Asp Leu Gly

115 120 125

Ser Asp Gly Arg Leu Leu Arg Gly Tyr Glu Gln Tyr Ala Tyr Asp Gly

130 135 140

Lys Asp Tyr Leu Ala Leu Asn Glu Asp Leu Arg Ser Trp Thr Ala Ala

145 150 155 160

Asp Thr Ala Ala Gln Ile Ser Lys Arg Lys Cys Glu Ala Ala Asn Val

165 170 175

Ala Glu Gln Arg Arg Ala Tyr Leu Glu Gly Thr Cys Val Glu Trp Leu

180 185 190

His Arg Tyr Leu Glu Asn Gly Lys Glu Met Leu Gln Arg Ala Asp Pro

195 200 205

Pro Lys Thr His Val Thr His His Pro Val Phe Asp Tyr Glu Ala Thr

210 215 220

Leu Arg Cys Trp Ala Leu Gly Phe Tyr Pro Ala Glu Ile Ile Leu Thr

225 230 235 240

Trp Gln Arg Asp Gly Glu Asp Gln Thr Gln Asp Val Glu Leu Val Glu

245 250 255

Thr Arg Pro Ala Gly Asp Gly Thr Phe Gln Lys Trp Ala Ala Val Val

260 265 270

Val Pro Ser Gly Glu Glu Gln Arg Tyr Thr Cys His Val Gln His Glu

275 280 285

Gly Leu Pro Glu Pro Leu Met Leu Arg Trp Lys Gln Ser Ser Leu Pro

290 295 300

Thr Ile Pro Ile Met Gly Ile Val Ala

305 310

<210> 5

<211> 338

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 5

Met Val Val Met Ala Pro Arg Thr Leu Phe Leu Leu Leu Ser Gly Ala

1 5 10 15

Leu Thr Leu Thr Glu Thr Trp Ala Gly Ser His Ser Met Arg Tyr Phe

20 25 30

Ser Ala Ala Val Ser Arg Pro Gly Arg Gly Glu Pro Arg Phe Ile Ala

35 40 45

Met Gly Tyr Val Asp Asp Thr Gln Phe Val Arg Phe Asp Ser Asp Ser

50 55 60

Ala Cys Pro Arg Met Glu Pro Arg Ala Pro Trp Val Glu Gln Glu Gly

65 70 75 80

Pro Glu Tyr Trp Glu Glu Glu Thr Arg Asn Thr Lys Ala His Ala Gln

85 90 95

Thr Asp Arg Met Asn Leu Gln Thr Leu Arg Gly Tyr Tyr Asn Gln Ser

100 105 110

Glu Ala Ser Ser His Thr Leu Gln Trp Met Ile Gly Cys Asp Leu Gly

115 120 125

Ser Asp Gly Arg Leu Leu Arg Gly Tyr Glu Gln Tyr Ala Tyr Asp Gly

130 135 140

Lys Asp Tyr Leu Ala Leu Asn Glu Asp Leu Arg Ser Trp Thr Ala Ala

145 150 155 160

Asp Thr Ala Ala Gln Ile Ser Lys Arg Lys Cys Glu Ala Ala Asn Val

165 170 175

Ala Glu Gln Arg Arg Ala Tyr Leu Glu Gly Thr Cys Val Glu Trp Leu

180 185 190

His Arg Tyr Leu Glu Asn Gly Lys Glu Met Leu Gln Arg Ala Asp Pro

195 200 205

Pro Lys Thr His Val Thr His His Pro Val Phe Asp Tyr Glu Ala Thr

210 215 220

Leu Arg Cys Trp Ala Leu Gly Phe Tyr Pro Ala Glu Ile Ile Leu Thr

225 230 235 240

Trp Gln Arg Asp Gly Glu Asp Gln Thr Gln Asp Val Glu Leu Val Glu

245 250 255

Thr Arg Pro Ala Gly Asp Gly Thr Phe Gln Lys Trp Ala Ala Val Val

260 265 270

Val Pro Ser Gly Glu Glu Gln Arg Tyr Thr Cys His Val Gln His Glu

275 280 285

Gly Leu Pro Glu Pro Leu Met Leu Arg Trp Lys Gln Ser Ser Leu Pro

290 295 300

Thr Ile Pro Ile Met Gly Ile Val Ala Gly Leu Val Val Leu Ala Ala

305 310 315 320

Val Val Thr Gly Ala Ala Val Ala Ala Val Leu Trp Arg Lys Lys Ser

325 330 335

Ser Asp

<210> 6

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic sequence

<400> 6

Gly Gly Gly Gly Ser

1 5

<210> 7

<211> 10

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic sequence

<400> 7

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 8

<211> 15

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic sequence

<400> 8

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 9

<211> 20

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic sequence

<400> 9

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser

20

Claims (74)

1. A therapeutic compound comprising:

i) a specific targeting moiety selected from the group consisting of:

a) a donor-specific targeting moiety that, for example, preferentially binds to a donor target; or

b) A tissue-specific targeting moiety that, for example, preferentially binds to a target tissue of a subject; and

ii) an effector binding/modulating moiety selected from:

(a) an immune cell inhibitory molecule binding/modulating moiety (ICIM binding/modulating moiety);

(b) an immunosuppressive immune cell binding/modulating moiety (IIC binding/modulating moiety); or

(c) An effector binding/modulating moiety as part of a therapeutic compound that promotes an immunosuppressive local microenvironment (SM binding/modulating moiety), for example by providing a substance proximal to a target that inhibits or minimizes the attack of the target's immune system.

2. The therapeutic compound of claim 1 wherein the effector binding/modulating moiety directly binds to and activates an inhibitory receptor.

3. The therapeutic compound of claim 2, wherein the effector binding/modulating moiety is an inhibitory immune checkpoint molecule.

4. The therapeutic compound of claim 3, wherein the effector binding/modulating moiety is expressed by an immune cell.

5. The therapeutic compound of claim 4, wherein the immune cells promote an undesired immune response.

6. The therapeutic compound of claim 4, wherein the immune cell causes a disease condition.

7. The therapeutic compound of claim 1, wherein the ability of a therapeutic molecule to agonize binding/modulation of the bound molecule by the effector is greater, e.g., 2, 5, 10, 100, 500, or 1,000 times greater, when the therapeutic compound is bound to a target by the targeting moiety than the ability of a therapeutic molecule to agonize binding/modulation of the bound molecule by the effector when the therapeutic compound is not bound to a target by the targeting moiety.

8. The therapeutic compound of claim 1, wherein the ability of the therapeutic molecule to agonize the effector binding/modulating the bound molecule when the therapeutic compound is bound to a target by the targeting moiety is greater than, e.g., 10, 100, 500, or 1,000 times greater than the ability of a therapeutic molecule to agonize the effector binding/modulating the bound molecule when the therapeutic compound is not bound to a target by the targeting moiety.

9. The therapeutic compound of claim 1 wherein there is significant systemic agonism of the molecule bound by the effector binding/modulating moiety at a therapeutically effective dose of the therapeutic compound.

10. The therapeutic compound of claim 1 wherein agonism of the molecule bound by the effector binding/modulating moiety occurs substantially only at the target site bound by the targeting moiety at a therapeutically effective dose of the therapeutic compound.

11. The therapeutic compound of claim 1, wherein the targeting moiety comprises an anti-MAdCAM antibody and the effector binding/modulating moiety comprises an anti-PD-1 antibody.

12. The therapeutic compound of claim 1 wherein binding of the effector binding/modulating moiety to its cognate ligand inhibits binding of an endogenous anti-ligand to the cognate ligand of the effector binding/modulating moiety by less than 60, 50, 40, 30, 20, 10, or 5%.

13. The therapeutic compound of claim 1, wherein binding of the effector binding/modulating moiety to the cognate ligand results in a substantial absence of antagonism of the cognate ligand of the effector binding/modulating molecule.

14. The therapeutic compound of claim 1, wherein the effector binding/modulating moiety comprises an ICIM binding/modulating moiety.

15. The therapeutic compound of any one of claims 14, wherein the effector binding/modulating moiety comprises an ICIM binding/modulating moiety comprising an inhibitory immune checkpoint molecule ligand molecule.

16. The therapeutic compound of any one of claims 15, wherein the inhibitory immune molecule anti-ligand molecule comprises a PD-L1 molecule.

17. The therapeutic compound of claim 14, wherein the ICIM is wherein the inhibitory immune molecule anti-ligand molecule is conjugated to a cognate inhibitory immune checkpoint molecule selected from PD-1, KIR2DL4, LILRB1, LILRB, or CTLA-4.

18. The therapeutic compound of claim 17, wherein the ICIM is an antibody.

19. The therapeutic compound of claim 18, wherein the ICIM comprises an antibody that binds to PD-1, KIR2DL4, LILRB1, LILRB, or CTLA-4.

20. The therapeutic compound of claim 19, wherein the antibody is an antibody that binds to PD-1.

21. The therapeutic compound of claim 19, wherein the antibody is an antibody that binds to PD-1 and is a PD-1 agonist.

22. The therapeutic compound of claim 19, wherein the antibody is an antibody that binds to PD-1 and is a PD-1 agonist when tethered to a target site.

23. The therapeutic compound of claim 1, wherein the effector binding/modulating moiety comprises an ICIM binding/modulating moiety comprising a functional antibody molecule directed against a cell surface inhibitory molecule.

24. The therapeutic compound of claim 1, wherein the cell surface inhibitory molecule is an inhibitory immune checkpoint molecule.

25. The compound of claim 24, wherein the inhibitory immune checkpoint molecule is selected from PD-1, KIR2DL4, LILRB1, LILRB2, CTLA-4, or from table 1.

26. The therapeutic compound of claim 1, further comprising a second effector binding/modulating moiety.

27. The therapeutic compound of claim 26, wherein the second effector binding/modulating moiety binds to a different target than the effector binding/modulating moiety.

28. The therapeutic compound of claim 26 or 27, wherein the second effector binding/modulating moiety comprises an IIC binding/modulating moiety.

29. The therapeutic compound of claim 26 or 27, wherein the second effector binding/modulating moiety comprises an SM binding/modulating moiety.

30. The therapeutic compound of claim 1 wherein the effector binding/modulating moiety comprises an IIC binding/modulating moiety.

31. The therapeutic compound of claim 1 wherein the effector binding/modulating portion comprises an IIC binding/modulating portion that increases, recruits or accumulates immunosuppressive immune cells at the target site.

32. The therapeutic compound of claim 1, wherein the effector binding/modulating moiety comprises a cell surface molecule binding agent that binds or specifically binds to a cell surface molecule on an immunosuppressive immune cell.

33. The therapeutic compound of claim 1, wherein the effector binding/modulating moiety comprises a cell surface molecule ligand molecule that binds or specifically binds to a cell surface molecule on an immunosuppressive immune cell.

34. The therapeutic compound of claim 1, wherein the effector binding/modulating moiety comprises an antibody molecule that binds to a cell surface molecule on an immunosuppressive immune cell.

35. The therapeutic compound of any one of claims 31-34, wherein the immunosuppressive immune cells comprise T regulatory cells, such as Foxp3+ CD25+ T regulatory cells.

36. The therapeutic compound of claim 1 wherein the effector binding/modulating moiety comprises an SM binding/modulating moiety.

37. The therapeutic compound of claim 36 wherein the SM binding/modulating moiety promotes an immunosuppressive local microenvironment.

38. The therapeutic compound of claim 36, wherein the SM binding/modulating moiety comprises a CD39 molecule.

39. The therapeutic compound of claim 36, wherein the SM binding/modulating moiety comprises a CD73 molecule.

40. The therapeutic compound of claim 36 wherein the SM binding/modulating moiety comprises an anti-CD 39 molecule.

41. The therapeutic compound of claim 36, wherein the SM binding/modulating moiety comprises an anti-CD 73 antibody molecule.

42. The therapeutic compound of claim 1, wherein the compound has the formula from N-terminus to C-terminus:

r1- -connecting sub-region A- -R2 or R3- -connecting sub-region B- -R4,

wherein the content of the first and second substances,

r1, R2, R3 and R4 each independently comprise an effector binding/modulating moiety, such as 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.

43. The therapeutic compound of claim 42, wherein either of linker region A and linker region B comprises an Fc region.

44. The therapeutic compound of claim 42, wherein one of R1 and R2 is an anti-PD-1 antibody and one of R1 and R2 is an anti-MAdCAM antibody.

45. The therapeutic compound of claim 42, wherein one of R1 is an anti-PD-1 antibody and one R2 is an anti-MAdCAM antibody.

46. The therapeutic compound of claim 42 wherein one of R1 is an anti-MAdCAM antibody and one R2 is an anti-PD-1 antibody.

47. The therapeutic compound of claim 42, wherein one of R3 and R4 is an anti-PD-1 antibody and one of R3 and R4 is an anti-MAdCAM antibody.

48. The therapeutic compound of claim 42, wherein one of R3 is an anti-PD-1 antibody and one R4 is an anti-MAdCAM antibody.

49. The therapeutic compound of claim 42 wherein one of R3 is an anti-MAdCAM antibody and one R4 is an anti-PD-1 antibody.

50. The therapeutic compound of any one of claims 44-49, wherein the linker is absent.

51. The therapeutic compound of any one of claims 44-49, wherein the linker is an Fc region.

52. The therapeutic compound of any one of claims 44-49, wherein the linker is a glycine/serine linker.

53. The therapeutic compound of any one of claims 44-49, wherein the PD-1 antibody is a PD-1 agonist.

54. The therapeutic compound of claim 44 wherein:

r1 and R3 independently comprise a functional anti-PD-1 antibody molecule (agonist of PD-1); and R2 and R4 independently comprise a specific targeting moiety, such as an scFv molecule directed against a tissue antigen.

55. The therapeutic compound of any one of claims 52-53, wherein:

r1 and R3 independently comprise a specific targeting moiety, such as an anti-tissue antigen antibody; and R2 and R4 independently comprise a functional anti-PD-1 antibody molecule (an agonist of PD-1).

56. The therapeutic compound of any one of the preceding claims, wherein the therapeutic compound comprises a PD-1 agonist.

57. The therapeutic compound of any one of the preceding claims, wherein the targeting moiety comprises a recognition moiety that binds to MAdCAM.

58. The therapeutic compound of any one of the preceding claims, wherein the targeting moiety comprises an antibody that binds or specifically binds to MAdCAM.

59. The therapeutic compound of any one of the preceding claims, wherein the targeting moiety comprises an antibody that binds or specifically binds to MAdCAM and the effector binding/modulating moiety comprises an antibody that binds to PD-1.

60. A method of treating a subject having an inflammatory bowel disease, the method comprising administering to the subject a therapeutic compound of any one of claims 1-59 to treat the inflammatory bowel disease.

61. The method of claim 60, wherein the subject having inflammatory bowel disease has Crohn's disease.

62. The method of claim 60, wherein the subject having inflammatory bowel disease has ulcerative colitis.

63. A method of treating a subject having autoimmune hepatitis, the method comprising administering to the subject a therapeutic compound of any one of claims 1-59 to treat the autoimmune hepatitis.

64. A method of treating primary sclerosing cholangitis, the method comprising administering to the subject a therapeutic compound according to any one of claims 1-59 to treat the primary sclerosing cholangitis.

65. A method of treating type 1 diabetes, the method comprising administering to the subject a therapeutic compound according to any one of claims 1-59 to treat the type 1 diabetes.

66. A method of treating a transplant subject comprising administering to the subject a therapeutically effective amount of the therapeutic compound of any one of claims 1-59, thereby treating a transplant (recipient) subject.

67. A method of treating GVHD in a subject having transplanted donor tissue, comprising administering to the subject a therapeutically effective amount of the therapeutic compound of any one of claims 1-59.

68. A method of treating a patient having, at risk of having, or at increased risk of having an autoimmune disorder, comprising administering a therapeutically effective amount of the therapeutic compound of any one of claims 1-59, thereby treating the subject.

69. A nucleic acid molecule encoding the therapeutic compound of any one of claims 1-59.

70. A vector comprising the nucleic acid of claim 69.

71. A cell comprising the nucleic acid of claim 59 or the vector of claim 70.

72. A method of making a therapeutic compound comprising culturing the cell of claim 71 to make the therapeutic compound.

73. A method of making a nucleic acid sequence encoding the therapeutic compound of claims 1-59, the method comprising

a) Providing a vector comprising a sequence encoding a targeting moiety and inserting into the vector sequence encoding an effector binding/modulating moiety to form a sequence encoding a therapeutic compound; or

b) Providing a vector comprising a sequence encoding an effector binding/modulating moiety and inserting into the vector sequence encoding a targeting moiety to form a sequence encoding a therapeutic compound,

thereby preparing a sequence encoding a therapeutic compound.

74. The method of any one of claims 60-73, wherein the subject has an autoimmune disorder and the therapeutic compound does not comprise an autoantigenic peptide or polypeptide characteristic of the autoimmune disorder, e.g., does not comprise a peptide or polypeptide to which the subject's autoantibodies are directed.