WO2018075462A1 - Compositions et procédés de traitement et de prévention d'une lésion associée à une greffe - Google Patents

Compositions et procédés de traitement et de prévention d'une lésion associée à une greffe Download PDF

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WO2018075462A1
WO2018075462A1 PCT/US2017/056898 US2017056898W WO2018075462A1 WO 2018075462 A1 WO2018075462 A1 WO 2018075462A1 US 2017056898 W US2017056898 W US 2017056898W WO 2018075462 A1 WO2018075462 A1 WO 2018075462A1
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inhibitor
complement
transplant
amino acids
seq
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PCT/US2017/056898
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English (en)
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Stephen Tomlinson
Carl ADKINSON
Xue-zhong YU
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Medical University Of South Carolina
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/436—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having oxygen as a ring hetero atom, e.g. rapamycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/57—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone
    • A61K31/573—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone substituted in position 21, e.g. cortisone, dexamethasone, prednisone or aldosterone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/675—Phosphorus compounds having nitrogen as a ring hetero atom, e.g. pyridoxal phosphate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • A61K38/13—Cyclosporins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca

Definitions

  • Tx organ or tissue transplantation
  • toxicity of immunosuppressive drugs is a serious concern since they can cause organ damage, metabolic dysfunction, cancer, and an increase in susceptibility to infection.
  • An important goal of Tx research is the development of strategies to minimize immunosuppression.
  • One approach being investigated to achieve this is the targeted delivery of immunosuppressive drugs to allografts, which by increasing drug bioavailability permits lower dosing and a reduction in systemic levels of immunosuppression.
  • transplantation is an accepted surgical approach for face or limb replacement, and Tx of composite tissue is required since such injuries involve multiple tissues.
  • Reconstructive surgery involving vascularized composite (VC) allotransplantation (VCA) is an emerging field, with about 150-200 procedures having been performed since the first successful hand transplant in 1998 (Dubernard et al., 1999, Lancet, 353(9161): 1315-1320).
  • tissue transplantation such as VCA
  • VCA tissue transplantation
  • An increased concern for this type of Tx is the toxicity of immunosuppressive drugs that can cause organ damage, metabolic dysfunction, cancer, and an increase in susceptibility to infection. These toxicities are applicable for all transplant patients, and an important goal in all Tx research, is the development of strategies to minimize immunosuppression.
  • graft rejection is principally dependent on T cells, there are other immune factors that can increase graft antigenicity leading to a strengthening of the rejection response.
  • IRI ischemia reperfusion injury
  • BDI brain death induced injury
  • the present invention provides a composition treating or preventing an injury associated with transplantation.
  • the present invention provides a composition treating or preventing an injury associated with transplantation.
  • composition comprises a targeted inhibitor molecule wherein the targeted inhibitor molecule comprises a targeting portion and an inhibitor portion.
  • the molecule inhibits the complement pathways.
  • the composition comprises a sub-therapeutic dose of an immunosuppressant agent.
  • the targeting portion comprises a CR2 protein or fragment thereof.
  • the inhibitor portion comprises at least one selected from the group consisting of FH, MCP, DAF, Cny, MAp44, CD59, and CR1.
  • the immunosuppressant agent comprises an agent selected from the group consisting of cyclosporine A, azathioprine, a corticosteroid including prednisone, and methylprednisolone, cyclophosphamide, FK506, and an mTOR inhibitor selected from a group consisting of rapamycin, sirolimus, and everolimus.
  • the present invention describes a method of treating or preventing an injury associated with transplantation wherein complement is activated.
  • the method comprises administering to a subject a therapeutically effective amount of a therapeutic agent.
  • the therapeutic agent comprises a targeted inhibitor molecule comprising a targeting portion and an inhibitor portion, wherein the molecule inhibits complement activation.
  • the transplant is a vascularized composite allograft, heart transplant, kidney transplant, liver transplant, lung transplant, pancreas transplant, intestine transplant, thymus transplant, musculoskeletal graft, cornea graft, skin graft, heart valve graft, nerves graft or vein graft.
  • the transplant is an allogeneic hematopoietic stem cell transplantation.
  • the targeting portion comprises CR2 or fragment thereof.
  • the inhibitor portion comprises at least one selected from the group consisting of FH, MCP, DAF, Cny, MAp44, CD59, and CR1.
  • method further comprises administering to the subject a sub-therapeutic amount of an immunosuppressant agent.
  • the immunosuppressant agent comprises an agent selected from the group consisting of cyclosporine A, azathioprine, a corticosteroid including prednisone, and
  • methylprednisolone cyclophosphamide
  • FK506 an mTOR inhibitor selected from a group consisting of rapamycin, sirolimus, and everolimus.
  • the subject has graft versus host disease. In one embodiment, the subject has an ischemia reperfusion injury of the transplant or at risk for developing an ischemia reperfusion injury of the transplant.
  • the present invention comprises a method of treating or preventing an injury associated with a transplant comprising administering to a transplant a therapeutically effective amount of a therapeutic agent.
  • the therapeutic agent comprises a targeted inhibitor molecule comprising a targeting portion and an inhibitor portion, wherein the molecule inhibits complement pathways
  • Figure 2A through Figure 2C is a set of images depicting IgM and C3d deposition in vascularized allografts isolated from mice 48 h after transplantation.
  • Figure 2B illustrates the representative immunohistochemistry images.
  • Figure 3 A and Figure 3B is a set of images depicting the effects of complement inhibition on the infiltration of neutrophils and macrophages into grafts.
  • Figure 4 is a set of images depicting effect of complement inhibition on the expression of P-selectin in grafts. All grafts were isolated from WT recipient mice with PBS (control) or CR2-crry treatment, CSaR ⁇ ' CSaR " " or C3 _/" recipient mice at 48 hours after transplantation. Expression of P-selectin was examined by
  • Figure 6A through Figure 6C is a set of images that depicts the combined CR2-Crry and subtherapeutic cyclosporine A treatment delays graft rejection and reduced T cell infiltration.
  • Figure 6B depicts the representative immunohistochemistry images of sections obtained from grafts 7 days post
  • Figure 7A and Figure 7B is a set of images depicting the combined CR2- Crry and CsA therapy reducing splenic Tel cell populations.
  • FIG. 7B demonstrate a significant reduction in overall number of CD8 + CXCR3 + cells in the combined therapy group, as compared to control and single therapy CR2-Crry and CsA groups.
  • CD4 + CXCR3 + T cells were significantly reduced in the combined group, as compared to Tx control. While there was an overall trend to reduced total numbers between combined and CsA alone groups, this did not reach statistical significance.
  • n 3 in each group).
  • Figure 8A through Figure 8C depicts exemplary CR2-FH constructs.
  • Figure 8A illustrates a CR2-FH expression plasmid.
  • Figure 8B illustrates a CR2-FH protein with signal peptide for an expression plasmid.
  • Figure 8C illustrates a mature CR2-FH protein.
  • Figure 9 depicts the amino acid sequence of human CR2 (referred to as
  • Figure 10 depicts the amino acid sequence of an exemplary human CR2- FH construct (referred to as SEQ NO: 2).
  • Figure 11 depicts exemplary amino acid sequence of mouse CR2 (SEQ ID NO: 1
  • Figure 12 depicts an amino acid sequence of an exemplary mouse CR2-
  • FH construct (SEQ ID NO: 4).
  • Figure 13 depicts C3d deposition at GVHD target organs.
  • Figure 14 depicts a structure of CR2-FH and a working hypothesis for
  • Figure 15 depicts that deficiency of FB but not Clq/MLB in that host ameliorates GVHD.
  • Figure 23 depicts that host alternative complement pathway depletion or systematic CR2-FH treatment effectively suppresses GVHD while preserving GVL.
  • Figure 16 depicts mechanisms for GVHD attenuation caused by host alternative complement pathway deficiency.
  • Figure 17 depicts the results of the in vitro characterization of Map44- CR2.
  • Figure 18, comprising Figure 18A and Figure 18B depicts the results of experiments evaluating the therapy of CR2-Crry in combination with subtherapeutic rapamic.
  • Subtherapeutic rapamycin and acute C inhibition synergise to inhibit the development of allograft vasculopathy.
  • Figure 18 A Quantification of luminal occlusion.
  • Figure 19 comprising Figure 19A through Figure 19D depicts the results of example experiments demonstrating the assessment of IRI damage in vascularized composite allografts from recipients of either living donor or brain dead donor grafts that had either been perfused with UW or UW augmented with CR2-Crry .
  • Histological quantification of injury in skin (Figure 19A) and muscle (Figure 19C) of grafts isolated at 48 hours post-transplant. Results are expressed as mean ⁇ SD; n 5 for all groups. ## p ⁇ 0.05 BD vs LD, **p ⁇ 0.05 BD vs BD+CR2-Crry. Representative histological images of skin (Figure 19C) and muscle ( Figure 19D).
  • Figure 21 depicts an exemplary nucleic acid sequence and amino acid sequence for mouse CR2-Crry.
  • the present invention is directed to compositions and methods for treating or preventing injury associated with tissue transplantation.
  • the invention provides for a reduced required dose of immunosuppression to prevent organ/tissue rejection or vasculopathy.
  • the present invention provides compositions and methods for treatment, inhibition, prevention or reduction of the inflammatory responses associated with the complement signaling.
  • the present invention may treat or prevent injuries such as donor brain death induced injuries (BDI) and ischemia-reperfusion injuries (IRI) that occur is tissue/organ transplants such as vascular composite allografts (VCA) and injuries resulting from graft-versus-host disease (GVHD) that can develop following allogeneic hematopoietic stem cell transplantation (all-HSCT).
  • the invention is related to compositions and methods affecting signaling associated with complement and products of complement activation thereof associated with transplants particularly associated with VCA and all-HSCT.
  • a potential problem in the translation of a complement inhibitor strategy to the clinic is the immunosuppressive effect of systemic complement inhibition, especially important in a Tx setting where the patient is immunocompromised.
  • complement has important roles in homeostatic and physiological functions such immune complex catabolism, clearance of dead and dying cells, tissue repair, modulation of adaptive immunity, neuroregenerative processes and host defense.
  • Other important concerns regarding the use of systemic complement inhibition relate to efficacy and biodistribution.
  • An approach to alleviate the concerns of systemic inhibition described herein specifically targets complement inhibition to sites of complement activation. In this approach, a fragment of C receptor 2 (CR2) that recognizes the C3d activation product is linked to a complement inhibitor.
  • CR2 C receptor 2
  • an element means one element or more than one element.
  • conjugated refers to covalent attachment of one molecule to a second molecule.
  • a “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal's health continues to deteriorate.
  • terapéuticaally effective amount refers to a sufficient amount of an agent to provide the desired biological result. That result can be reduction and/or alleviation of a sign, symptom, or cause of a disease or disorder, or any other desired alteration of a biological system. An appropriate effective amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.
  • fusion protein refers to two or more peptides, polypeptides, or proteins operably linked to each other.
  • “Graft” refers to a cell, tissue, organ or otherwise any biological compatible substrate for transplantation.
  • mammals include, but are not limited to, farm animals, sport animals, pets, primates, mice and rats.
  • the individual is human.
  • the individual is an individual other than human.
  • inhibitor means to suppress or block an activity or function relative to a control value.
  • the activity is suppressed or blocked by 10% compared to a control value, more preferably by 50%, and even more preferably by 95%.
  • nucleotide sequence encoding an amino acid sequence includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence.
  • the phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).
  • polypeptide As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds.
  • a protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence.
  • Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types.
  • Polypeptides include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified
  • polypeptides derivatives, analogs, fusion proteins, among others.
  • the polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.
  • pharmaceutically acceptable refers to agents that, within the scope of sound medical judgment, are suitable for use in contact with tissues of human beings and/or animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • subject refers to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein.
  • patient, subject or individual is a human.
  • sub-therapeutic as used herein means a treatment at a dose known to be less than what is known to induce a therapeutic effect.
  • terapéutica as used herein means a treatment and/or prophylaxis.
  • a therapeutic effect is obtained by suppression, remission, or eradication of a disease state.
  • therapeutic agent use herein refers to any agent that has a therapeutic effect and/or elicits a desired biological and/or pharmacological effect, when administered to a subject.
  • an agent is considered to be a therapeutic agent if its administration to a relevant population is statistically correlated with a desired or beneficial therapeutic outcome in the population, whether or not a particular subject to whom the agent is administered experiences the desired or beneficial therapeutic outcome.
  • terapéuticaally effective amount means an amount that is sufficient, when administered to a population suffering from or susceptible to a disease, disorder, and/or condition in accordance with a therapeutic dosing regimen, to treat the disease, disorder, and/or condition (e.g., host versus graft disease).
  • a therapeutically effective amount is one that reduces the incidence and/or severity of, and/or delays onset of, one or more symptoms of the disease, disorder, and/or condition.
  • a therapeutically effective amount does not in fact require successful treatment be achieved in a particular individual.
  • a therapeutically effective amount may be that amount that provides a particular desired pharmacological response in a significant number of subjects when administered to patients in need of such treatment. It is specifically understood that particular subjects may, in fact, be “refractory” to a “therapeutically effective amount.” To give but one example, a refractory subject may have a low bioavailability such that clinical efficacy is not obtainable.
  • reference to a therapeutically effective amount may be a reference to an amount as measured in one or more specific tissues (e.g., a tissue affected by the disease, disorder or condition) or fluids (e.g., blood, saliva, serum, sweat, tears, urine, etc.).
  • a therapeutically effective agent may be formulated and/or administered in a single dose. In some embodiments, a therapeutically effective agent may be formulated and/or administered in a plurality of doses, for example, as part of a dosing regimen.
  • tissue transplantation refers to a transfer of tissue or tissue components from an external source into a recipient (host) individual.
  • the external source is an organism (e.g., a living, brain dead, recently dead, or dead organism).
  • the external source is an ex vivo or in vitro system.
  • a “vector” is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell.
  • vectors are known in the art including, but not limited to, linear
  • vector includes an autonomously replicating plasmid or a virus.
  • the term should also be construed to include non-plasmid and non- viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like.
  • viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.
  • ranges throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.
  • the invention describes a therapeutic composition and method for treating or preventing injury associated with transplantation.
  • the invention reduces the required dose of immunosuppression to prevent organ/tissue rejection or vasculopathy.
  • the composition comprises a targeted complement inhibitor and an immunosuppressant agent.
  • the complement inhibitor is a composite molecule comprised of a targeting portion and an inhibitor portion wherein the composite molecule targets complement signaling.
  • the complement-targeting portion comprises CR2.
  • the composition comprises a complement-targeted inhibitor and an immunosuppressant agent.
  • the composition comprises a sub-therapeutic level of the immunosuppressant agent.
  • the sub-therapeutic level of the immunosuppressant agent is a level of the immunosuppressant agent that has little to no efficacy if used alone.
  • the immunosuppressant agent comprises cyclosporine A.
  • the present invention significantly prolongs graft viability without the need for traditional anti -rejection therapies including but not limited to therapeutic levels of immunosuppression therapy.
  • the present invention is based in part on the discovery that CR2-Crry composition efficaciously reduce activation of the complement pathways reducing inflammation in many indications including atherosclerosis, multiple sclerosis and IRI. Accordingly, the present invention relates to compositions and methods for improving the viability of vascular composite allografts (VCA), reducing transplant damage, and reducing the risk to the recipient relating to the side effects around graft rejection including those involving the complement pathway.
  • VCA vascular composite allografts
  • the present invention relates to a composition used to treat a subject that is a recipient of a transplant.
  • the composition modulates complement signaling.
  • the composition of the present invention comprises a composite molecule comprising a targeting portion and an inhibitor portion.
  • the targeting portion directs the molecule to sites of injury or inflammation, and the inhibitor portion inhibit complement signaling.
  • the targeting portion is CR2.
  • the inhibitor portion is selected from a list comprising but not limited to Factor H (FH), Crry, DAF, MCP, MAp44, CD59, and CR1.
  • the composition is the combination of the composite molecule in addition to a sub-therapeutic amount of an immunosuppressant agent.
  • the immunosuppressant therapeutic agent selected from a list of agents comprising cyclosporine A azathioprine, corticosteroids including prednisone, and
  • methylprednisolone cyclophosphamide
  • FK506 cyclophosphamide
  • mTOR inhibitors including rapamycin, sirolimus, and everolimus.
  • the complement-targeting portion of the herein described composition is CR2.
  • the CR2 portion described herein comprises a CR2 or a fragment thereof.
  • CR2 is a protein encoded by the CR2 gene and is involved in the complement system. It binds to iC3b (inactive derivative of C3b), C3dg, or C3d, B cells have CR2 receptors on their surfaces, allowing the complement system to play a role in B-cell activation and maturation.
  • CR2 consists of an extracellular portion consisting of 15 or 16 repeating units known as short consensus repeats (SCRs).
  • Amino acids 1-20 comprise the leader peptide
  • amino acids 23-82 comprise SCR1
  • amino acids 91-146 comprise SCR2
  • amino acids 154-210 comprise SCR3, amino acids 215-271 comprise SCR4.
  • the active site (C3dg binding site) is located in SCR 1-2 (the first 2 N-terminal SCRs). SCR units are separated by short sequences of variable length that serve as spacers. It is understood that any number of SCRs containing the active site can be used.
  • the construct contains the 4 N-terminal SCR units.
  • the construct includes the first two N-terminal SCRs. In another
  • the construct includes the first three N-terminal SCRs.
  • the CR2 portion comprises the first two N-terminal SCR domains of CR2. In some embodiments, the CR2 portion comprises the first three N-terminal SCR domains of CR2. In some embodiments, the CR2 portion comprises the first four N-terminal SCR domains of CR2. In some embodiments, the CR2 portion comprises (and in some embodiments consists of or consists essentially of) at least the first two N-terminal SCR domains of CR2, including for example at least any of the first 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 SCR domains of CR2.
  • a homologue of a CR2 protein or a fragment thereof includes proteins which differ from a naturally occurring CR2 (or CR2 fragment) in that at least one or a few amino acids have been deleted (e.g., a truncated version of the protein, such as a peptide or fragment), inserted, inverted, substituted and/or derived (e.g., by glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitation, amidation and/or addition of glycosylphosphatidyl inositol).
  • a CR2 homologue has an amino acid sequence that is at least about 70% identical to the amino acid sequence of a naturally occurring CR2 (e.g., SEQ ID NO: 1, or SEQ ID NO:3), for example at least about any of 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of a naturally occurring CR2 (e.g., SEQ ID NO: l, or SEQ ID NO:3).
  • a naturally occurring CR2 e.g., SEQ ID NO: 1, or SEQ ID NO:3
  • a CR2 homologue or a fragment thereof preferably retains the ability to bind to a naturally occurring ligand of CR2 (e.g., C3d or other C3 fragments with CR2 -binding ability).
  • the CR2 homologue (or fragment thereof) may have a binding affinity for C3d that is at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of that of CR2 (or a fragment thereof) .
  • the CR2 portion comprises at least the first two N- terminal SCR domains of a human CR2, such as a CR2 portion having an amino acid sequence containing at least amino acids 23 through 146 of the human CR2 (SEQ ID NO: 1).
  • the CR2 portion comprises at least the first two SCR domains of human CR2 having an amino acid sequence that is at least about any of 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to amino acids 23 through 146 of the human CR2 (SEQ ID NO: l).
  • amino acid sequence that is at least about, for example, 95% identical to a reference sequence (such as SEQ ID NO: 1 or SEQ ID NO: 3) is intended that the amino acid sequence is identical to the reference sequence except that the amino acid sequence may include up to five point alterations per each 100 amino acids of the reference sequence. These up to five point alterations may be deletions, substitutions, additions, and may occur anywhere in the sequence, interspersed either individually among amino acids in the reference sequence or in one or more continuous groups within the reference sequence.
  • the CR2 portion comprises part or all of the ligand binding sites of the CR2 protein. In some embodiments, the CR2 portion further comprises sequences required to maintain the three dimensional structure of the binding site.
  • Ligand binding sites of CR2 can be readily determined based on the crystal structures of CR2, such as the human and mouse CR2 crystal structures disclosed in U.S. Patent Application Publication No. 2004/0005538.
  • the CR2 portion comprises the B strand and B-C loop of SCR2 of CR2.
  • the CR2 portion comprises a site on strand B and the B-C loop of CR2 SCR comprising the segment G98-G99-Y100-K101-I102-R103-G104-S105-T106-P107- Y108 with respect to SEQ ID NO: 1.
  • the CR2 portion comprises a site on the B strand of CR2 SCR2 comprising position Kl 19 with respect to SEQ ID NO: l . In some embodiments, the CR2 portion comprises a segment comprising V149- F150-P151-L152, with respect to SEQ ID NO: 1. In some embodiments, the CR2 portion comprises a segment of CR2 SCR2 comprising T120-N121-F122. In some embodiments, the CR2 -inhibitor molecule has two or more of these sites.
  • the CR2 portion comprises a portion comprising G98-G99-Y100-K101- I102-R103-G104-5105-T106-P107-Y108 and Kl 19 with respect to SEQ ID NO: 1. Other combinations of these sites are also contemplated.
  • Inhibitor Portion comprises a portion comprising G98-G99-Y100-K101- I102-R103-G104-5105-T106-P107-Y108 and Kl 19 with respect to SEQ ID NO: 1. Other combinations of these sites are also contemplated.
  • the molecules described herein in some embodiments comprise an inhibitor portion comprising a complement modulator, such as a complement inhibitor.
  • complement inhibitor refers to any compound, composition, or protein that reduces or eliminates complement activity.
  • the reduction in complement activity may be incremental (e.g., a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%), or 90% reduction in activity) or complete.
  • a complement inhibitor can inhibit complement activity by at least 10 (e.g., at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 or greater) % in a standard in vitro red blood cell hemolysis assay or an in vitro CH50eq assay.
  • the CH50eq assay is a method for measuring the total classical complement activity in serum.
  • This test is a lytic assay, which uses antibody- sensitized erythrocytes as the activator of the classical complement pathway and various dilutions of the test serum to determine the amount required to give 50% lysis (CH50).
  • CH50 50% lysis
  • the percent hemolysis can be determined, for example, using a spectrophotometer.
  • the CH50eq assay provides an indirect measure of terminal complement complex (TCC) formation, since the TCC themselves are directly responsible for the hemolysis that is measured.
  • TCC terminal complement complex
  • the assay is well known and commonly practiced by those of skill in the art. Briefly, to activate the classical complement pathway, undiluted serum samples (e.g., human serum samples) are added to microassay wells containing the antibody-sensitized erythrocytes to thereby generate TCC. Next, the activated sera are diluted in microassay wells, which are coated with a capture reagent (e.g., an antibody that binds to one or more components of the TCC). The TCC present in the activated samples bind to the monoclonal antibodies coating the surface of the microassay wells. The wells are washed and, to each well, is added a detection reagent that is detectably labeled and recognizes the bound TCC. The detectable label can be, e.g., a fluorescent label or an enzymatic label. The assay results are expressed in CH50 unit equivalents per milliliter (CH50 U Eq/mL).
  • a capture reagent e.g.,
  • the complement inhibitor described herein in some embodiments is a specific inhibitor of the lectin pathway. In some embodiments, the complement inhibitor is a specific inhibitor of the alternative pathway. In some embodiments, the complement inhibitor is a specific inhibitor of the classical pathway.
  • the complement inhibitor is a soluble or membrane-bound protein such as, for example, membrane cofactor protein (MCP), decay accelerating factor (DAF/CD55), CD59, mouse complement receptor 1-related gene/protein y (Crry), human complement receptor 1 (CR1) or factor H, or Factor I, or an antibody specific for a component of a complement pathway such as, for example, eculizumab (an anti-CS antibody marketed under the trade name Soliris®), pexelizumab (the antigen-binding fragment of eculizumab), an anti-factor B antibody (such as the monoclonal antibody 1379 produced by ATCC Deposit No.
  • MCP membrane cofactor protein
  • DAF/CD55 decay accelerating factor
  • CD59 mouse complement receptor 1-related gene/protein y
  • Crry mouse complement receptor 1-related gene/protein y
  • CR1 or factor H or Factor I
  • an antibody specific for a component of a complement pathway such as, for example, eculizumab (an anti-CS antibody
  • a complement inhibitor may be a small molecule or a linear or cyclic peptide such as, for example, compstatin, N- acetylaspartylglutamic acid (NAAGA), and the like.
  • the complement inhibitor is selected from the group consisting of: an anti-C5 antibody, an Eculizumab, an pexelizumab, an anti-C3b antibody, an anti-C6 antibody, an anti-C7 antibody, an anti- factor B antibody, an anti- factor D antibody, and an anti-properdin antibody, a human membrane co factor protein (MCP), a human decay accelerating factor (DAF), a mouse decay accelerating factor (DAF), a human CD59, a mouse CD59, a mouse CD59 isoform B, a mouse Crry, a human CR1, a Factor I, a human factor H, a mouse factor H, and a biologically active fragment of any the preceding.
  • MCP human membrane co factor protein
  • DAF human decay accelerating factor
  • DAF mouse decay accelerating factor
  • MCP membrane cofactor protein
  • CD46 refers to a widely distributed C3b/C4b-binding cell surface glycoprotein which inhibits complement activation on host cells and serves as a cofactor for the factor I-mediated cleavage of C3b and C4b, including ho mo logs thereof. T.J. Oglesby et al., J. Exp. Med. (1992) 175: 1547-1551.
  • MCP belongs to a family known as the regulators of complement activation ("RCA"). Family members share certain structural features, comprising varying numbers of short consensus repeat (SCR) domains, which are typically between 60 and 70 amino acids in length.
  • SCR short consensus repeat
  • MCP comprises four SCRs, a serine/threonine/proline-enriched region, an area of undefined function, a transmembrane hydrophobic domain, a cytoplasmic anchor and a cytoplasmic tail. It is understood that species and strain variations exist for the disclosed peptides,
  • polypeptides, and proteins, and that human MCP or biologically active fragments thereof encompass all species and strain variations.
  • SEQ ID NO: 5 represents the full-length human MCP amino acid sequence (see, e.g., UniProtKB/Swiss-Prot. Accession No. P15529).
  • Amino acids 1-34 correspond to the signal peptide
  • amino acids 35-343 correspond to the extracellular domain
  • amino acids 344-366 correspond to the transmembrane domain
  • amino acids 367-392 correspond to the cytoplasmic domain.
  • amino acids 35-96 correspond to SCR 1
  • amino acids 97-159 correspond to SCR 2
  • amino acids 160-225 correspond to SCR 3
  • amino acids 226-285 correspond to SCR 4
  • amino acids 302- 326 correspond to the serine/threonine-rich domain. It is understood that species and strain variations exist for the disclosed peptides, polypeptides, and proteins, and that
  • MCP or biologically active fragments thereof encompass all species and strain variations.
  • biologically active fragment of MCP refers to any soluble fragment lacking both the cytoplasmic domain and the transmembrane domain, including fragments comprising, consisting essentially of or consisting of 1, 2, 3, or 4 SCR domains, with or without the serine/threonine-rich domain, having some or all of the complement inhibitory activity of the full-length human MCP protein.
  • the complement inhibitor portion comprises full-length human MCP (amino acids 35-392 of SEQ ID NO:5), the extracellular domain of human MCP (amino acids 35-343 of SEQ ID NO:5), or SCRs 1-4 of human MCP (amino acids 35-285 of SEQ ID NO:5).
  • Decay accelerating factor also referred to as CD55 (DAF/CD55) (SEQ ID NO:6 and SEQ ID NO:7), is an -70 kiloDalton (kDa) membrane-bound glycoprotein which inhibits complement activation on host cells.
  • DAF comprises several approximately 60 amino acid repeating motifs termed short consensus repeats (SCR).
  • DAF decay accelerating factor
  • kDa seventy kilodalton
  • SCR short consensus repeat
  • GPI glycosylphosphatidylinositol
  • DAF protects the cell surface from complement activation by dissociating membrane-bound C3 convertases that are required to cleave complement protein C3 and to amplify the complement cascade. DAF prevents assembly or accelerates decay of both the C3- and C5-convertases of the alternative and classical complement pathways.
  • SEQ ID NO: 6 represents the full-length human DAF amino acid sequence
  • SEQ ID NO:7 represents the full-length mouse DAF amino acid sequence (see, e.g., UniProtKB/Swiss-Prot. Accession No. Q61475).
  • amino acids 1-34 correspond to the signal peptide
  • amino acids 35-353 appear in the mature protein
  • amino acids 354-381 are removed from the polypeptide after translation.
  • amino acids 35-96 correspond to SCR 1
  • amino acids 96- 160 correspond to SCR 2
  • amino acids 161- 222 correspond to SCR 3
  • amino acids 223-285 correspond to SCR 4
  • amino acids 287-353 correspond to the O-glycosylated serine/threonine-rich domain.
  • the GPI anchor is attached to human DAF at a serine at position 353.
  • amino acids 1-34 correspond to the signal peptide
  • amino acids 35-362 appear in the mature protein
  • amino acids 363-390 are removed from the polypeptide after translation.
  • amino acids 35-96 correspond to SCR 1
  • amino acids 97-160 correspond to SCR 2
  • amino acids 161-222 correspond to SCR 3
  • amino acids 223-286 correspond to SCR 4
  • amino acids 288-362 correspond to the O-glycosylated serine/threonine-rich domain.
  • the GPI anchor is attached to mouse DAF at a serine at position 362. It is understood that species and strain variations exist for the disclosed peptides, polypeptides, and proteins, and that DAF or biologically active fragments thereof encompass all species and strain variations.
  • biologically active fragment of DAF refers to any fragment of DAF lacking a GPI anchor and/or the amino acid to which it is attached (i.e., Ser-353), including any fragments of the full- length DAF protein comprising, consisting essentially of or consisting of 1, 2, 3, or 4 SCR domains, with or without the O-glycosylated serine/threonine-rich domain, having some or all the complement inhibitory activity of the full-length DAF protein.
  • CD59 refers to a membrane-bound 128 amino acid glycoprotein that potently inhibits the membrane attack complex (MAC) of complement.
  • CD59 acts by binding to the C8 and/or C9 components of the MAC during assembly, ultimately preventing incorporation of the multiple copies of C9 required for complete formation of the osmolytic pore at the heart of the MAC.
  • CD59 is both N- and O-glycosylated.
  • the N-glycosylation comprises primarily bi- or tri-antennary structures with and without lactosamine and outer arm fucose residues, with variable sialylation present at some sites.
  • CD59 is anchored in the cell membrane by a glycosylphosphatidylinositol (“GPI”) anchor, which is attached to an asparagine at amino acid 102.
  • GPI glycosylphosphatidylinositol
  • Soluble forms of CD59 have been produced, but they generally have low functional activity in vitro, particularly in the presence of serum, suggesting that unmodified sCD59 has little or no therapeutic efficacy. See, e.g., S. Meri et al.,
  • SEQ ID NO:8 represents the full-length human CD59 amino acid sequence (see, e.g., UniProtKB/Swiss-Prot. Accession No. P13987);
  • SEQ ID NO:9 represents the full-length mouse CD59 sequence, isoform A (see, e.g., UniProtKB/Swiss- Prot. Accession No. 055186);
  • SEQ ID NO: 10 represents the full-length mouse CD59 sequence, isoform B (see, e.g., UniProtKB/SwissProt. Accession No. P58019).
  • amino acids 1-25 of SEQ ID NO:8 correspond to the leader peptide
  • amino acids 26-102 of SEQ ID NO: 8 correspond to the mature protein
  • amino acids 103-128 of SEQ ID NO:8 are removed after translation.
  • the GPI anchor is attached to CD59 at an asparagine at position 102 of SEQ ID NO: 8.
  • amino acids 1-23 of SEQ ID NO: 9 correspond to the leader peptide
  • amino acids 24-96 of SEQ ID NO: 9 correspond to the mature protein
  • amino acids 97-123 of SEQ ID NO: 9 are removed after translation.
  • the GPI anchor is attached to CD59 at a serine at position 96 of SEQ ID NO: 9.
  • amino acids 1-23 of SEQ ID NO: 10 correspond to the leader peptide
  • amino acids 24-104 of SEQ ID NO: 10 correspond to the mature protein
  • amino acids 105-129 of SEQ ID NO: 10 are removed after translation.
  • the GPI anchor is attached to CD59 at an asparagine at position 104 of SEQ ID NO: 10. It is understood that species and strain variations exist for the disclosed peptides, polypeptides, and proteins, and that CD59 or biologically active fragments thereof encompass all species and strain variations.
  • biologically active fragment of human CD59 refers to any fragment of human CD59 lacking a GPI anchor and/or the amino acid to which it is attached (i.e., Asn-102), including any fragments of the full-length human CD59 protein having some or all the complement inhibitory activity of the full-length CD59 protein; and the term “biologically active" fragment of mouse CD59 refers to any fragment of mouse CD59 isoform A or isoform B lacking a GPI anchor and/or the amino acid to which it is attached (i.e., Ser-96 of isoform A, or Asp- 104 of isoform B), including any fragments of either full-length mouse CD59 protein isoform having some or all the complement inhibitory activity of the full-length CD59 protein.
  • mae complement receptor 1 -related gene/protein y refers to a membrane-bound mouse glycoprotein that regulates complement activation, including homologs thereof. Crry regulates complement activation by serving as a cofactor for complement factor I, a serine protease which cleaves C3b and C4b deposited on host tissue. Crry also acts as a decay- accelerating factor, preventing the formation of C4b2a and C3bBb, the amplification convertases of the complement cascade.
  • SEQ ID NO: 11 represents the full-length mouse Crry protein amino acid sequence.
  • Amino acids 1-40 correspond to the leader peptide
  • amino acids 41-483 of SEQ ID NO: 11 correspond to the mature protein, comprising amino acids 41-405 of SEQ ID NO : 11 , corresponding to the extracellular domain
  • amino acids 427-483 of SEQ ID NO: 11 corresponding to the cytoplasmic domain.
  • amino acids 83-143 of SEQ ID NO: 11 correspond to SCR 1
  • amino acids 144-205 of SEQ ID NO: 11 correspond to SCR 2
  • amino acids 206-276 of SEQ ID NO: 11 correspond to SCR 3
  • amino acids 277-338 of SEQ ID NO: 11 correspond to SCR 4
  • amino acids 339- 400 of SEQ ID NO: 11 correspond to SCR 5. It is understood that species and strain variations exist for the disclosed peptides, polypeptides, and proteins, and that mouse Crry protein or biologically active fragments thereof encompasses all species and strain variations.
  • biologically active fragment of mouse Crry protein refers to any soluble fragment of mouse Crry lacking the transmembrane domain and the cytoplasmic domain, including fragments comprising, consisting essentially of or consisting of 1, 2, 3, 4, or 5 SCR domains, including any fragments of the full-length mouse Crry protein having some or all the complement inhibitory activity of the full- length Crry protein.
  • the biologically active fragment of mouse Crry comprises amino acids 85-403 of SEQ ID NO: 11.
  • complement receptor 1 refers to a human gene encoding a protein of 2039 amino acids, with a predicted molecular weight of 220 kilodaltons (“kDa”), including homologs thereof.
  • the gene is expressed principally on erythrocytes, monocytes, neutrophils, and B cells, but is also present on some T lymphocytes, mast cells, and glomerular podocytes.
  • CR1 protein is typically expressed at between 100 and 1000 copies per cell. CR1 is the main system for processing and clearance of complement-opsonized immune complexes.
  • CR1 negatively regulates the complement cascade, mediates immune adherence and phagocytosis, and inhibits both the classic and alternative complement pathways.
  • the full-length CR1 protein comprises a 42 amino acid signal peptide, an extracellular domain of 1930 amino acids, a 25 amino acid transmembrane domain, and a 43 amino acid C-terminal cytoplasmic domain.
  • the extracellular domain of CR1 has 25 potential N-glycosylation signal sequences, and comprises 30 short consensus (“SCR") domains, also known as complement control protein (CCP) repeats, or sushi domains, each 60 to 70 amino acids long.
  • SCR short consensus
  • CCP complement control protein
  • the 30 SCR domains are further grouped into four longer regions termed long homologous repeats ("LHRs"), each encoding approximately 45 kDa segments of the CR1 protein, designated LHR-A, -B, -C, and -D.
  • LHRs long homologous repeats
  • the first three comprise seven SCR domains each, while LHR-D comprises 9 SCR domains.
  • the active sites on the extracellular domain of CR1 protein include a C4b-binding site with lower affinity for C3b in SCRs 1-4 comprising amino acids 42-295, a C3b-binding site with lower affinity for C4b in SCRs 8-11 comprising amino acids 490-745, a C3b-binding site with lower affinity for C4b in SCRs 15-18 comprising amino acids 940-1196, and a Clq-binding site in SCRs 22-28 comprising amino acids 1394-1842.
  • SEQ ID NO: 12 represents the full-length human CR1 amino acid sequence
  • amino acids 1-41 correspond to the signal peptide
  • amino acids 42-2039 correspond to the mature protein, comprising amino acids 42-1971, corresponding to the extracellular domain, amino acids 1972-1996, corresponding to the transmembrane domain, and amino acids 1997-2039, corresponding to the cytoplasmic domain.
  • amino acids 42-101 correspond to SCR 1
  • 102-163 correspond to SCR2
  • amino acids 164-234 correspond to SCR3
  • amino acids 236-295 correspond to SCR4
  • amino acids 295-355 correspond to SCR5
  • amino acids 356-418 correspond to SCR6
  • amino acids 419-489 correspond to SCR7
  • amino acids 491-551 correspond to SCR8
  • amino acids 552- 613 correspond to SCR9
  • amino acids 614-684 correspond to SCRIO
  • amino acids 686-745 correspond to SCR1
  • amino acids 745-805 correspond to SCR12
  • amino acids 806-868 correspond to SCR13
  • amino acids 869-939 correspond to SCR 14
  • amino acids 941-1001 correspond to SCR15
  • amino acids 1002-1063 correspond to SCR16
  • amino acids 1064-1134 correspond to SCR17
  • amino acids 1136-1195 correspond to SCR18
  • amino acids 1195-1255 correspond to SCR 19
  • amino acids 1256-1318 correspond to SCR 20
  • CR1 protein or biologically active fragments thereof encompass all species and strain variations.
  • biologically active fragment of CR1 protein refers to any soluble fragment of CR1 lacking the
  • transmembrane domain and the cytoplasmic domain including fragments comprising, consisting essentially of or consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 SCR domains, including any fragments of the full-length CR1 protein having some or all the complement inhibitory activity of the full-length CR1 protein.
  • complement factor H As used herein, the term "complement factor H,” “factor H,” or “FH” refers to complement factor H, a single polypeptide chain plasma glycoprotein, including homologs thereof.
  • the protein is composed of 20 conserved short consensus repeat (SCR) domains of approximately 60 amino acids, arranged in a continuous fashion like a string of beads, separated by short linker sequences of 2-6 amino acids each.
  • SCR conserved short consensus repeat
  • Factor H binds to C3b, accelerates the decay of the alternative pathway C3-convertase (C3bBb), and acts as a cofactor for the proteolytic inactivation of C3b.
  • C3bBb alternative pathway C3-convertase
  • proteolysis by factor I results in the cleavage and inactivation of C3b.
  • Factor H has at least three distinct binding domains for C3b, which are located within SCRs 1-4, SCRs 5- 8, and SCRs 19-20. Each domain binds to a distinct region within the C3b protein: the N- terminal sites bind to native C3b; the second site, located in the middle region of factor H, binds to the C3c fragment and the site located within SCR19 and 20 binds to the C3d region.
  • factor H also contains binding sites for heparin, which are located within SCR 7, SCRs 5-12, and SCR 20 of factor Hand overlap with those of the C3b binding sites. Structural and functional analyses have shown that the domains for the complement inhibitory activity of factor H are located within the first four N-terminal SCR domains.
  • SEQ ID NO: 13 represents the full-length human factor H amino acid sequence (see, e.g., UniProtKB/Swiss-Prot. Accession No. P08603); SEQ ID NO: 14 represents the full-length mouse factor H amino acid sequence (see, e.g.,
  • amino acids 1-18 of SEQ ID NO: 13 correspond to the signal peptide
  • amino acids 19-1231 of SEQ ID NO: 13 correspond to the mature protein.
  • amino acids 21- 80 of SEQ ID NO: 13 correspond to SCR 1
  • amino acids 85-141 of SEQ ID NO: 13 correspond to SCR 2
  • amino acids 146-205 of SEQ ID NO: 13 correspond to SCR 3
  • amino acids 210-262 of SEQ ID NO: 13 correspond to SCR 4
  • amino acids 267-320 of SEQ ID NO: 13 correspond to SCR 5.
  • amino acids 1- 18 of SEQ ID NO: 14 correspond to the signal peptide
  • amino acids 19-1234 of SEQ ID NO: 14 correspond to the mature protein.
  • amino acids 19-82 of SEQ ID NO: 14 correspond to SCR 1
  • amino acids 83-143 of SEQ ID NO: 14 correspond to SCR 2
  • amino acids 144-207 of SEQ ID NO: 14 correspond to SCR 3
  • amino acids 208-264 of SEQ ID NO: 14 correspond to SCR 4
  • amino acids 265-322 of SEQ ID NO: 14 correspond to SCR 5.
  • biologically active fragment of factor H refers to any portion of a factor H protein having some or all the complement inhibitory activity of the full-length factor H protein, and includes, but is not limited to, factor H fragments comprising SCRs 1-4, SCRs 1-5, SCRs 1-8, SCRs 1-18, SCRs 19-20, or any homolog of a naturally-occurring factor H or fragment thereof, as described in detail below.
  • the biologically active fragment of factor H has one or more of the following properties: (1) binding to C-reactive protein (CRP), (2) binding to C3b, (3) binding to heparin, (4) binding to sialic acid, (5) binding to endothelial cell surfaces, (6) binding to cellular integrin receptor, (7) binding to pathogens, (8) C3b co-factor activity, (9) C3b decay- acceleration activity, and (10) inhibiting the alternative complement pathway.
  • CRP C-reactive protein
  • SEQ ID NO: 15 represents the amino acid sequence for mannose-binding lectin-associated protein of 44kDa (MAp44).
  • MAp44 is an alternatively spliced product encoded by the MASP1 gene.
  • MAp44 is an inhibitor of lectin pathway activation.
  • the inhibitor portion of the targeted molecule described herein comprises a complement inhibitor or biologically active fragment thereof.
  • the complement inhibitor is selected from the group consisting of human MCP, human DAF, mouse DAF, human CD59, mouse CD59 isoform A, mouse CD59 isoform B, mouse Crry protein, human CRl, human factor H, or mouse factor H, a Factor I, MAp44 or a biologically active fragment thereof.
  • the inhibitor portion comprises full-length human MCP (SEQ ID NO:5).
  • the complement inhibitor portion of the targeting construct comprises a biologically active fragment of human MCP (SEQ ID NO:5).
  • the biologically active fragment of human MCP is selected from the group consisting of SCRs 1-4 (amino acids 35-285 of SEQ ID NO:5), SCRs 1-4 plus the serine/threonine-rich domain (amino acids 35-326 of SEQ ID NO:5), and the extracellular domain of MCP (amino acids 35-343 of SEQ ID NO:5).
  • the inhibitor portion comprises full-length human
  • the inhibitor portion comprises a biologically active fragment of human DAF (SEQ ID NO:6).
  • the biologically active fragment of human DAF is selected from the group consisting of SCRs 1-4 (amino acids 25-285 of SEQ ID NO:6) and SCRs 1-4 plus the O- glycosylated serine/threonine-rich domain (amino acids 25-353 of SEQ ID NO:6).
  • the inhibitor portion comprises full-length mouse DAF (SEQ ID NO:7).
  • the inhibitor portion comprises a biologically active fragment of mouse DAF.
  • the biologically active fragment of mouse DAF is selected from the group consisting of SCRs 1-4 (amino acids 35-286 of SEQ ID NO:7) and SCRs 1-4 plus the O- glycosylated serine/threonine-rich domain (amino acids 35-362 of SEQ ID NO:7).
  • the inhibitor portion comprises full-length human
  • the inhibitor portion comprises a biologically active fragment of human CD59 (SEQ ID NO:8).
  • the biologically active fragment of human CD59 comprises the extracellular domain of human CD59 lacking its GPI anchor (amino acids 26-101 of SEQ ID NO:8).
  • the inhibitor portion comprises full-length mouse CD59, isoform A (SEQ ID NO:9).
  • the inhibitor portion comprises a biologically active fragment of mouse CD59, isoform A (SEQ ID NO:9).
  • the biologically active fragment of mouse CD59, isoform A comprises the extracellular domain of mouse CD59, isoform A lacking its GPI anchor (amino acids 24-95 of SEQ ID NO:9).
  • the inhibitor portion comprises full-length mouse CD59, isoform B (SEQ ID NO: 10).
  • the c inhibitor portion comprises a biologically active fragment of mouse CD59, isoform B (SEQ ID NO: 10).
  • the biologically active fragment of mouse CD59, isoform B comprises the extracellular domain of mouse CD59, isoform lacking its GPI anchor (amino acids 24- 103 of SEQ ID NO: 10).
  • the inhibitor portion comprises full-length mouse Crry protein (SEQ ID NO: 11). In some embodiments, the inhibitor portion comprises a biologically active fragment of mouse Crry protein (SEQ ID NO: l 1). In some embodiments, the biologically active fragment of mouse Crry protein is selected from the group consisting of SCRs 1-5 (amino acids 41-400 of SEQ ID NO: 11) and the extracellular domain of mouse Crry protein (amino acids 41-405 of SEQ ID NO: 11). In one embodiment, the inhibitor portion comprises the biologically active fragment of mouse Crry comprising amino acids 85-403 of SEQ ID NO: 11.
  • the inhibitor portion comprises full-length human CR1 protein (SEQ ID NO: 12).
  • the t inhibitor portion comprises a biologically active fragment of human CR1 protein (SEQ ID NO: 12).
  • the biologically active fragment of human CRl protein is selected from the group consisting of SCRs 1-4 (amino acids 42-295 of SEQ ID NO: 12), SCRs 1-10 (amino acids 42-684 of SEQ ID NO: 12), SCRs 8-11 (amino acids 490-745 of SEQ ID NO: 12), SCRs 15-18 (amino acids 940-1196 of SEQ ID NO: 12), and SCRs 22-28 (amino acids 1394-1842 of SEQ ID NO: 12).
  • the inhibitor portion comprises full-length human (SEQ ID NO: 13) or mouse (SEQ ID NO: 14) factor H. In some embodiments, the inhibitor portion comprises a biologically active fragment of human (SEQ ID NO: 13) or mouse (SEQ ID NO: 14) factor H.
  • the biologically active fragment of human factor H is selected from the group consisting of SCRs 1-4 (amino acids 21-262 of SEQ ID NO: 13), SCRs 1-5 of factor H (amino acids 21-320 of SEQ ID NO: 13), SCRs 1-8 of factor H (amino acids 21-507 of SEQ ID NO: 13), and SCRs 1-18 of factor H (amino acids 21-1104 of SEQ ID NO: 13).
  • the biologically active fragment of mouse factor H is selected from the group consisting of SCRs 1-4 (amino acids 19-264 of SEQ ID NO: 14), SCRs 1-5 of factor H (amino acids 19-322 of SEQ ID NO: 14), SCRs 1-8 of factor H (amino acids 19- 507 of SEQ ID NO: 14), and SCRs 1-18 of factor H (amino acids 19-1109 of SEQ ID NO: 14).
  • the biologically active fragment of human (SEQ ID NO: 13) or mouse (SEQ ID NO: 14) factor H comprises (and in some embodiments consists of or consists essentially of) at least the first four N-terminal SCR domains of factor H, including for example, at least any of the first 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or more N-terminal SCR domains of factor H.
  • the inhibitor portion comprises MAp44 (SEQ ID NO: 15). In some embodiments, the inhibitor portion comprises a biologically active fragment of MAp44 (SEQ ID NO: 15).
  • the inhibitor portion of the targeted molecules is a homolog of any of the complement inhibitors described herein or a biologically active fragment thereof.
  • Homologs of the complement inhibitors include proteins which differ from a naturally occurring complement inhibitor (or biologically-active fragment thereof) in that at least one or a few, but not limited to one or a few, amino acids have been deleted (e.g., a truncated version of the protein, such as a peptide or fragment), inserted, inverted, substituted and/or derivatized (e.g., by glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitation, amidation and/or addition glycosylphosphatidyl inositol).
  • homologue of a complement inhibitor may have an amino acid sequence that is at least about 70% identical to the amino acid sequence of a naturally complement inhibitor (e.g., SEQ ID NOs:5-12), for example at least about any of 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%), 97%), 98%), or 99% identical to the amino acid sequence of a naturally occurring complement inhibitor (e.g., SEQ ID NOs:5-15).
  • a naturally complement inhibitor e.g., SEQ ID NOs:5-12
  • Amino acid sequence identity can be determined in various ways, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGNTM (DNAST AR) software.
  • BLAST BLAST-2
  • ALIGN ALIGN
  • MEGALIGNTM DNAST AR
  • a homolog of complement inhibitor retains all the alternative complement pathway inhibitory activity of the complement inhibitor (or a biologically active fragment thereof) from which it is derived. In certain embodiments, the homolog of a complement inhibitor (or a biologically-active fragment thereof) retains at least about 50%, for example, at least about any of 60%, 70%, 80%, 90%, or 95% of the complement inhibition activity the complement inhibitor (or a biologically-active fragment thereof) from which is derived.
  • the inhibitor portion comprises an antibody (or an antigen binding fragment thereof) that binds to a complement component, e.g., a complement component selected from the group consisting of CI, Clq, Cis, C2, C2a, C3, C3a, C3b, C4, C4b, C5, C5a, C5b, C6, C7, C8, and C9.
  • a complement component selected from the group consisting of CI, Clq, Cis, C2, C2a, C3, C3a, C3b, C4, C4b, C5, C5a, C5b, C6, C7, C8, and C9.
  • the complement polypeptides to which the antibodies or antigen binding fragments thereof bind can be, in some embodiments, human polypeptides, e.g., human CI, Clq, Cls, C2, C2a, C3, C3a, C3b, C4, C4b, C5, C5a, C5b, C6, C7, C8, C9, factor B, factor D, or properdin polypeptides.
  • human polypeptides e.g., human CI, Clq, Cls, C2, C2a, C3, C3a, C3b, C4, C4b, C5, C5a, C5b, C6, C7, C8, C9, factor B, factor D, or properdin polypeptides.
  • the amino acid sequences for the foregoing complement proteins are well-known in the art as are methods for preparing the proteins or fragments thereof for use in preparing an antibody (or antigen-binding fragment thereof) specific for one or more of the
  • anti-complement protein antibodies which are suitable for incorporation into the targeted molecules described herein and for subsequent use in any of the methods described herein, are also well known in the art.
  • antibodies that bind to complement component C5 and inhibit the cleavage of C5 into fragments C5a and C5b include, e.g., eculizumab (Soliris®; Alexion Pharmaceuticals, Inc.,
  • Cheshire, CT and pexelizumab (Alexion Pharmaceuticals, Inc., Cheshire, CT). See, e.g., Kaplan (2002) Curr Opin Investig Drugs 3(7): 1017-23; Hill (2005) Clin Adv Hematol Oncol 3(11):849-50; Rother et al. (2007) Nature Biotechnol 25(11): 1256-1488; Whiss (2002) Curr Opin Investig Drugs 3(6):870-7; Patel et al. (2005) Drugs Today (Bare) 41(3): 165-70; and Thomas et al. (1996) Mol Immunol. 33(17- 18): 1389-401.
  • the anti-C5 antibody can bind to an epitope in the alpha chain of the human complement component C5 protein.
  • Antibodies that bind to the alpha chain of C5 are described in, for example, PCT application publication no. WO
  • the anti-C5 antibody can bind to an epitope in the beta chain of the human complement component C5 protein.
  • Antibodies that bind to the C5 beta chain are described in, e.g., Moongkamdi et al. (1982) Immunobiol 162:397; Moongkamdi et al. (1983) Immunobiol 165:323; and Mollnes et al. (1988) Scand 1 Immunol 28:307-312.
  • Antibodies that bind to C3b and, for example, inhibit the C3b convertase are also well known in the art.
  • PCT application publication nos. WO 2010/136311, WOb2009/056631, and WO 2008/154251 the disclosures of each of which are incorporated herein by reference in their entirety.
  • Antagonistic anti-C6 antibodies and anti-C7 antibodies have been described in, e.g., Brauer et al.(1996) Transplantation 61(4):S88-S94 and U.S. patent no. 5,679,345.
  • the inhibitor portion comprises an anti-factor B antibody (such as the monoclonal antibody 1379 produced by ATCC Deposit No. PTA- 6230).
  • Anti-factor B antibodies are also described in, e.g., Ueda et al. (1987) J Immunol 138(4): 1143-9; Tanhehco et al. (1999) Transplant Proc 31(5):2168-71; U.S. patent application publication nos. 20050260198 and 2008029911; and PCT publication no. WO 09/029669.
  • the inhibitor portion comprises an anti-factor D antibody, e.g., an antibody described in Pascual et al. (1990) 1 Immunol Methods 127:263-269; Sahu et al. (1993) Mol Immunol 30(7):679-684; Pascual et al. (1993) Eur 1 Immunol 23 : 1389-1392; Niemann et al. (1984) J Immunol 132(2):809-815; U.S. patent no. 7,439,331; or U.S. patent application publication no. 20080118506.
  • an anti-factor D antibody e.g., an antibody described in Pascual et al. (1990) 1 Immunol Methods 127:263-269; Sahu et al. (1993) Mol Immunol 30(7):679-684; Pascual et al. (1993) Eur 1 Immunol 23 : 1389-1392; Niemann et al. (1984) J Immunol 132(2):809
  • the inhibitor portion comprises an anti-properdin antibody.
  • Suitable anti-properdin antibodies are also well-known in the art and include, e.g., U.S. patent application publication nos. 20110014614 and PCT application publication no. W02009110918.
  • the inhibitor portion comprises an anti-MBL antibody.
  • Mannose-binding mannan-binding lectin a plasma protein, forms a complex with proteins known as MBL-associated serine proteases (MASPs).
  • MBL binds to several monosaccharides that are uncharacteristic of mammalian proteins, e.g., mannose, N-acetylglucosamine, N-acetylmannoseamine, L-fucose and glucose, whereas sialic acid and galactose are not bound.
  • MBL-MASP complex When the MBL-MASP complex binds to microorganisms, the proenzymic forms of the serine proteases are activated and mediate the activation of complement components C4 and C2, thereby generating the C3 convertase C4b2b and leading to opsonization by the deposition of C4b and C3b fragments.
  • MASP -2 has been shown to cleave C4 and C2, while MASP-1 may be responsible for direct cleavage of C3.
  • the functions of MASP-3 and MApl9 are less well understood. Studies have shown a clear link between low levels of MBL and opsonic deficiency, as well as clinical manifestations such as severe diarrhea, chronic hepatitis and HIV infection, and autoimmune disease.
  • Anti-mannan-binding lectin antibodies are known in the art (see, e.g., Pradhan et al. (2012) Rheumatol. Int. epublished September, 2012) and commercially available (AbCam).
  • the inhibitor portion comprises an anti-MASP antibody.
  • MASPs mannan-binding lectin-associated serine proteases
  • MASP-1, MASP-2 and MASP-3 mannan-binding lectin-associated serine protease- 1, -2 and -3
  • MASP-1 has a histidine loop structure of the type found in trypsin and trypsin-like serine proteases. MASP-1 has been found to be involved in complement activation by MBL. A cDNA clone encoding MASP-1 has been reported that encodes a putative leader peptide of 19 amino acids followed by 680 amino acid residues predicted to form the mature peptide. MASP-2 (MBL-associated serine protease 2) is a serine protease also similar in structure to CI r and CI s of the complement pathway. Like these, and contrary to MASP-1, it has no histidine loop structure of the type found in trypsin and trypsin-like serine proteases. It has been theorized that MASP-1 can cleave C3, generating C3b, which may be deposited on an activated cell or tissue surface
  • MASP-2 cleaves C4 and C2, giving rise to the C3 convertase, C4b2b (Thiel et al., Nature, 386:506-10 (1997)).
  • the MASP-2 protein comprises of a number of domains namely the CUB1, EGF, CUB2, CCP1, CCP2 and serine protease domains. It is believed that the domain responsible for association with MBL is situated in the N-terminus, whereas the serine protease domain is responsible for the serine protease activity of MASP-2.
  • sMAP also known as MApl9
  • MApl9 is a 19 kd is derived from the same gene as MASP-2, which lacks the serine protease domain and a major part of the A chain. Skjoedt et al., Immunobiology, 215:921-31 (2010). Recently, a third member of the family, MASP-3 was identified, which shares a high degree of homology with MASP-1, such that it appears that MASP-1 and MASP-3 are generated as a result of alternative splicing of primary mRNA transcripts.
  • MBL/MASP complex and their use for inhibiting the adverse effects of complement activation, such as ischemia-reperfusion injury, have been disclosed, for example, in WO04/075837; US 2009/0017031.
  • MBL/Ficolin Associated Protein (MAP- 1), which is present in low serum levels compared to MASP-1 and MASP-3, has been reported to function as a local lectin pathway specific complement inhibitor. Skjodt et al., Molecular Immunology, 47:2229-30 (2010). Accordingly, MAP-1 itself, or fragments of MAP-1, may be useful in the present invention as an inhibitor of MASP, and accordingly, as a lectin-pathway- specific inhibitor of complement activation.
  • the ficolin family of proteins are characterized by carbohydrate binding and opsonic activities, sharing a structure similar to MBL. Like MBL, the ficolins have been shown to associate with MASPs in serum and may mediate complement activation in response to
  • inhibitors of the ficolin family or functional fragments therof may be useful in the present invention as an inhibitor of MASPs and as a lectin-pathway specific inhibitor of complement activation.
  • the inhibitor portion comprises an antibody (or antigen binding fragment thereof) that specifically binds to a human complement component protein (e.g., human C5, C6, C7, C8, or C9).
  • a human complement component protein e.g., human C5, C6, C7, C8, or C9.
  • the terms "specific binding” or “specifically binds” refer to two molecules forming a complex (e.g., a complex between an antibody and a complement component protein) that is relatively stable under physiologic conditions. Typically, binding is considered specific when the association constant (Ka) is higher than 106 M-l.
  • an antibody can specifically bind to a C5 protein with a Ka of at least (or greater than) 106 (e.g., at least or greater than 107, 108, 109, 1010, 1011, 1012, 1013, 1014, or 1015 or higher) M-l.
  • Ka of at least (or greater than) 106
  • M-l M-l.
  • Examples of antibodies that specifically bind to a human complement component C5 protein are described in, e.g., U.S. patent no. 6,355,245 and PCT application publication no. WO 2010/015608.
  • Methods for determining whether an antibody binds to a protein antigen and/or the affinity for an antibody to a protein antigen are known in the art and described herein.
  • the binding of an antibody to a protein antigen can be detected and/or quantified using a variety of techniques such as, but not limited to, Western blot, dot blot, surface plasmon resonance method (e.g., BIAcore system; Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.), or enzyme-linked immunosorbent assay (ELISA) assays.
  • Western blot e.g., BIAcore system; Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.
  • ELISA enzyme-linked immunosorbent assay
  • the targeted molecule also includes an amino acid linker sequence linking the targeting portion and the inhibitor portion.
  • the CR2 portion and the inhibitor portion are non- covalently linked.
  • the two portions may be brought together by two interacting bridging proteins (such as biotin and streptavidin), each linked to a CR2 portion or the inhibitor portion.
  • the molecule described herein thus generally has the dual functions of binding to a CR2 ligand and inhibiting complement activation of the alternative pathway.
  • CR2 ligand refers to any molecule that binds to a naturally occurring CR2 protein, which include, but are not limited to, C3d, iC3b, C3dg, C3d, and cell-bound fragments of C3b that bind to the two N-terminal SCR domains of CR2.
  • the CR2-FH molecule may, for example, bind to a CR2 ligand with a binding affinity that is about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the CR2 protein.
  • Binding affinity can be determined by any method known in the art, including for example, surface plasmon resonance, calorimetry titration, ELISA, and flow cytometry.
  • the molecule has one or more of the following properties of CR2: (1) binding to C3d, (2) binding to iC3b, (3) binding to C3dg, (4) binding to cell-bound fragment(s) of C3b that bind to the two N-terminal SCR domains of CR2.
  • the molecule described herein is generally capable of inhibiting complement activation of complement activation (for example the alternative pathway, classical pathway, and/or lectin pathway).
  • the molecule may be a more potent complement inhibitor than the naturally occurring inhibitor protein.
  • the molecule has a complement inhibitory activity that is about any of 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 40, or more fold of that of the inhibitor protein.
  • the molecule has an EC50 of less than about any of 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, or 10 nM.
  • the molecule has an EC50 of about 5-60 nM, including for example any of 8-50 nM, 8-20 nM, 10-40 nM, and 20-30 nM. In some embodiments, the molecule has complement inhibitory activity that is about any of 50%, 60%, 70%, 80%, 90%, or 100% of that of the inhibitor protein.
  • Complement inhibition can be evaluated based on any methods known in the art, including for example, in vitro zymosan assays, assays for lysis of erythrocytes, antibody or immune complex activation assays, alternative pathway activation assays, classical pathway activation assays and mannan (lectin pathway) activation assays.
  • the molecule is a fusion protein.
  • the CR2 portion and the inhibitor portion are directly fused to each other.
  • the CR2 portion and the inhibitor portion are linked by an amino acid linker sequence.
  • Linking sequences can also comprise "natural" linking sequences found between different domains of complement factors.
  • VSVFPLE the linking sequence between the first two N-terminal short consensus repeat domains of human CR2
  • EEIF the linking sequence between the fourth and the fifth N-terminal short consensus repeat domains of human CR2
  • the order of CR2 portion and inhibitor portion in the fusion protein can vary.
  • the C-terminus of the CR2 portion is fused (directly or indirectly) to the N-terminus of the inhibitor portion of the molecule.
  • the N-terminus of the CR2 portion is fused (directly or indirectly) to the C- terminus of the inhibitor portion of the molecule.
  • the molecule is a CR2-FH fusion protein.
  • the molecule is a fusion protein.
  • Fusion protein used herein refers to two or more peptides, polypeptides, or proteins operably linked to each other.
  • the targeting portion and inhibitor portion are directly fused to each other.
  • the targeting portion and inhibitor portion are linked by an amino acid linker sequence.
  • linker sequences are known in the art, and include, for example, (Gly4Ser), (Gly4Ser)2, (Gly4Ser)3, (Gly3Ser)4, (SerGly4), (SerGly4)2, (SerGly4)3, and (SerGly4)4.
  • Linking sequences can also comprise "natural" linking sequences found between different domains of complement factors.
  • the order of targeting portion and inhibitor portion in the fusion protein can vary.
  • the C-terminus of the targeting portion is fused (directly or indirectly) to the N-terminus of the inhibitor portion of the targeting construct.
  • the N-terminus of the targeting portion is fused (directly or indirectly) to the C-terminus of the inhibitor portion of the molecule.
  • the molecule comprises a CR2 portion and an inhibitor portion linked via a chemical cross-linker.
  • Linking of the two portions can occur on reactive groups located on the two portions.
  • Reactive groups that can be targeted using a crosslinker include primary amines, sulfhydryls, carbonyls, carbohydrates, and carboxylic acids, or active groups that can be added to proteins.
  • chemical linkers include, but are not limited to, bismaleimidohexane, maleimidobenzoyl-N-hydroxysuccinimide ester, NHS-Esters-Maleimide crosslinkers such as SPDP, carbodiimide, glutaraldehyde, MBS, sulfo-MBS, SMPB, sulfo-SMPB,
  • GMBS sulfo-GMBS
  • EMCS sulfo-EMCS
  • imidoester crosslinkers such as DMA, DMP, DMS, DTBP, EDC and DTME.
  • the CR2 portion and the inhibitor portion are non- covalently linked.
  • the two portions may be brought together by two interacting bridging proteins (such as biotin and streptavidin), each linked to a CR2 portion or an inhibitor portion.
  • the molecule comprises two or more (same or different) CR2 portions described herein.
  • the molecule comprises two or more (same or different) inhibitor portions described herein. These two or more CR2 (or inhibitor) portions may be tandemly linked (such as fused) to each other.
  • the molecule (such a CR2 -inhibitor fusion protein) comprises a CR2 portion and two or more (such as three, four, five, or more) inhibitor portions. In some embodiments, the molecule (such a CR2 -inhibitor fusion protein) comprises an inhibitor portion and two or more (such as three, four, five, or more) CR2 portions. In some embodiments, the molecule (such a CR2 -inhibitor fusion protein) comprises two or more CR2 portions and two or more inhibitor portions.
  • an isolated molecule In some embodiments, there is provided an isolated molecule. In some embodiments, the molecules form dimers or multimers.
  • the CR2 portion and the inhibitor portion in the molecule can be from the same species (such as human or mouse), or from different species.
  • the CR2-inhibitor molecule comprises CR2Crry comprising the amino acid sequence of SEQ ID NO: 18.
  • An exemplary nucleotide sequence encoding CR2Crry is provided in SEQ ID NO: 17.
  • the CR2-inhibitor molecule comprises a homolog or biologically active fragment of
  • CR2Crry Homologs of the CR2Crry include proteins which differ from CR2Crry described herein (or biologically-active fragment thereof) in that at least one or a few, but not limited to one or a few, amino acids have been deleted (e.g., a truncated version of the protein, such as a peptide or fragment), inserted, inverted, substituted and/or derivatized (e.g., by glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitation, amidation and/or addition glycosylphosphatidyl inositol).
  • a truncated version of the protein such as a peptide or fragment
  • derivatized e.g., by glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitation, amidation and/or addition glycosylphosphatidyl inosito
  • homologue of CR2Crry may have an amino acid sequence that is at least about 70% identical to the amino acid sequence CR2Crry (e.g., SEQ ID NO: 18), for example at least about any of 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of CR2Crry (e.g., SEQ ID NO: 18).
  • Amino acid sequence identity can be determined in various ways, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGNTM (DNAST AR) software.
  • BLAST BLAST-2
  • ALIGN ALIGN
  • MEGALIGNTM DNAST AR
  • the CR2-inhibitor molecule comprises human CR2- FH comprising the amino acid sequence of SEQ ID NO: 2.
  • the CR2- inhibitor molecule comprises a homolog or biologically active fragment of human CR2- FH.
  • Homologs of human CR2-FH include proteins which differ from human CR2-FH described herein (or biologically-active fragment thereof) in that at least one or a few, but not limited to one or a few, amino acids have been deleted (e.g., a truncated version of the protein, such as a peptide or fragment), inserted, inverted, substituted and/or derivatized (e.g., by glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitation, amidation and/or addition glycosylphosphatidyl inositol).
  • homologue of human CR2-FH may have an amino acid sequence that is at least about 70% identical to the amino acid sequence human CR2-FH (e.g., SEQ ID NO: 2), for example at least about any of 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,
  • amino acid sequence identity can be determined in various ways, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or
  • MEGALIGNTM DNAST AR
  • the CR2-inhibitor molecule comprises mouse CR2- FH comprising the amino acid sequence of SEQ ID NO: 4.
  • the CR2- inhibitor molecule comprises a homolog or biologically active fragment of mouse CR2- FH.
  • Homologs of mouse CR2-FH include proteins which differ from mouse CR2-FH described herein (or biologically-active fragment thereof) in that at least one or a few, but not limited to one or a few, amino acids have been deleted (e.g., a truncated version of the protein, such as a peptide or fragment), inserted, inverted, substituted and/or derivatized (e.g., by glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitation, amidation and/or addition glycosylphosphatidyl inositol).
  • homologue of mouse CR2-FH may have an amino acid sequence that is at least about 70% identical to the amino acid sequence mouse CR2-FH (e.g., SEQ ID NO: 4), for example at least about any of 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of mouse CR2-FH (e.g., SEQ ID NO: 4).
  • Amino acid sequence identity can be determined in various ways, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or
  • MEGALIGNTM DNAST AR
  • the CR2-inhibitor molecules (or the two portions of the CR2 -inhibitor molecules) described herein may be made by chemical synthesis methods, or by linkage of a polynucleotide encoding the CR2 portion and a polynucleotide encoding the inhibitor portion (with or without a linker sequence), and introducing the resulting polynucleotide molecule in a vector for transfecting host cells that are capable of expressing the molecule. Chemical synthesis, especially solid phase synthesis, is preferred for short peptides or those containing unnatural or unusual amino acids such as D-Tyr, Ornithine, and the like. Recombinant procedures are preferred for longer polypeptides.
  • the molecule can be isolated in vitro by protein purification methods.
  • the molecule can also be provided "in situ" by introduction of a gene therapy system to the tissue of interest which then expresses the CR2-inhibitor fusion.
  • Recombinant DNA techniques for making a CR2-inhibitor fusion protein involves, in simplified form, taking the a CR2-inhibitor encoding polynucleotide, inserting it into an appropriate vector, inserting the vector into an appropriate host cell, and recovering or isolating the fusion protein produced thereby.
  • polynucleotides that encode a CR2-inhibitor molecule
  • a CR2-inhibitor fusion protein Such polynucleotide may also be used for delivery and expression of CR2-inhibitor.
  • a polynucleotide encoding a fusion protein comprising a CR2 portion comprising a CR2 or a fragment thereof and an inhibitor portion comprising an intact inhibitor molecule or a fragment thereof.
  • the polynucleotide also comprises a sequence encoding a signal peptide operably linked at the 5' end of the sequence encoding the CR2 -inhibitor fusion protein.
  • a linker sequence is used for linking the CR2 portion and the inhibitor portion.
  • the polynucleotide encodes a CR2 -inhibitor fusion protein.
  • expression vectors comprising a polynucleotide described herein for expression of the CR2-inhibitor fusion protein.
  • the expression vector can be used to direct expression of a CR2 -inhibitor fusion protein in vitro or in vivo.
  • the vector may include any element to establish a conventional function of a vector, for example, promoter, terminator, selection marker, and origin of replication.
  • the promoter can be constitutive or regulative, and is selected from, for example, promoters of genes for galactokinase (GALl), uridylyltransferase (GALT), epimerase (GAL 10), phosphoglycerate kinase (PGK), glyceraldehydes-3 -phosphate dehydrogenase (GPD), alcohol dehydrogenase (ADH), and the like.
  • GALl galactokinase
  • GALT uridylyltransferase
  • GAL 10 epimerase
  • PGK phosphoglycerate kinase
  • GPD glyceraldehydes-3 -phosphate dehydrogenase
  • ADH alcohol dehydrogenase
  • E. coli may be transformed using pBR322, a plasmid derived from an E. coli species (Mandel et al., J. Mol. Biol., 53 : 154 (1970)). Plasmid pBR322 contains genes for ampicillin and tetracycline resistance, and thus provides easy means for selection. Other vectors include different features such as different promoters, which are often important in expression.
  • plasmids pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden), pKK233-2 (Clontech, Palo Alto, Calif, USA), and pGEMl (Promega Biotech, Madison, Wis., USA), are all commercially available.
  • Other vectors that can be used in the present invention include, but are not limited to, pET21a (Studier et al., Methods Enzymol., 185: 60-89 (1990)), pRlT5, and pRlT2T (Pharmacia Biotechnology), and pB0475 (Cunningham et al., Science, 243 : 1330-1336 (1989); U.S. Pat. No.
  • Mammalian expression vectors may contain non-transcribed elements such as an origin of replication, promoter and enhancer, and 5' or 3' nontranslated sequences such as ribosome binding sites, a polyadenylation site, acceptor site and splice donor, and transcriptional termination sequences.
  • Promoters for use in mammalian expression vectors usually are for example viral promoters such as Polyoma, Adenovirus, HTLV, Simian Virus 40 (SV 40), and human cytomegalovirus (CMV).
  • Vectors can also be constructed using standard techniques by combining the relevant traits of the vectors described above.
  • host cells such as isolated cells, transient cell lines, and stable cell lines for expressing a CR2-inhibitor fusion protein.
  • the host cell may be prokaryotic or eukaryotes.
  • Exemplary prokaryote host cells include E. coli K12 strain 294 (ATCC No. 31446), E. coli B, E. coli XI 776 (ATCC No. 31537), E. coli W3110 (F-, gamma-, prototrophic/ ATCC No. 27325), bacilli such as Bacillus subtilis, and other enterobacteriaceae such as Salmonella typhimurium or Serratia marcesans, and various Pseudomonas species.
  • E. coli BL21 (Stratagene), which is deficient in the OmpT and Lon proteases, which may interfere with isolation of intact recombinant proteins, and useful with T7 promoter-driven vectors, such as the pET vectors.
  • Another suitable prokaryote is E. coli W3110 (ATCC No. 27325).
  • the peptides When expressed by prokaryotes the peptides typically contain an N-terminal methionine or a formyl methionine and are not glycosylated. In the case of fusion proteins, the N-terminal methionine or formyl methionine resides on the amino terminus of the fusion protein or the signal sequence of the fusion protein.
  • eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for fusion-protein-encoding vectors.
  • Saccharomyces cerevisiae is a commonly used lower eukaryotic host microorganism.
  • Others include Schizosaccharomyces pombe (Beach and Nurse, Nature, 290: 140 (1981); EP 139,383 published 2 May 1985); Kluyveromyces hosts (U.S. Pat. No. 4,943,529; Fleer et al., Bio/Technology, 9:968-975 (1991)) such as, e.g., K.
  • lactis (MW98-8C, CBS683, CBS4574; Louvencourt et al., J. Bacterid., 154(2): 737-742 (1983)), K. fragilis (ATCC 12,424), K. bulgaricus (ATCC No. 16,045), K. wickeramii (ATCC No. 24,178), K. waltii (ATCC No. 56,500), K. drosophilarum (ATCC No. 36,906; Van den Berg et al., Bio/Technology, 8: 135 (1990)), K. thermotolerans, and K.
  • Aspergillus hosts such as A. nidulans (Ballance et al., Biochem. Biophys. Res. Commun., 112:284-289 (1983); Tilburn et al., Gene, 26:205-221 (1983); Yelton et al., Proc. Natl. Acad. Sci. USA, 81 : 1470-1474 (1984)) and A. niger (Kelly and Hynes, EMBO J., 4:475- 479 (1985)).
  • Methyl otropic yeasts are suitable herein and include, but are not limited to, yeast capable of growth on methanol selected from the genera consisting of Hansenula, Candida, Kloeckera, Pichia, Saccharomyces, Torulopsis, and Rhodotorula.
  • yeast capable of growth on methanol selected from the genera consisting of Hansenula, Candida, Kloeckera, Pichia, Saccharomyces, Torulopsis, and Rhodotorula.
  • Host cells also include insect cells such as Drosophila S2 and Spodoptera Sf9, as well as plant cells.
  • Examples of useful mammalian host cell lines include, but are not limited to, HeLa, Chinese hamster ovary (CHO), COS-7, L cells, C127, 3T3, BHK, CHL-1, NSO, HEK293, WI38, BHK, C127 or MDCK cell lines.
  • Another exemplary mammalian cell line is CHL-1.
  • CHL-1 cells are derived from RPMI 7032 melanoma cells, a readily available human cell line. Cells suitable for use in this invention are commercially available from the ATCC.
  • the host cell is a non-human host cell. In some embodiment, the host cell is a CHO cell. In some embodiments, the host cell is a 293 cell.
  • the CR2-inhibitor molecules can be isolated by a variety of methods known in the art. In some embodiments, when the CR2-inhibitor molecule is a fusion protein secreted into the growth media, the molecule can be purified directly from the media. If the fusion protein is not secreted, it is isolated from cell lysates. Cell disruption can be done by any conventional method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents. The CR2 -inhibitor molecules can be obtained by various methods.
  • the CR2-inhibitor molecule can be purified by immunoaffinity chromatography using an antibody that recognizes the CR2 portion or an antibody that recognizes the inhibitor portion, or both.
  • an antibody recognizing the first two N-terminal SCR domains of CR2 is used for purifying the CR2- inhibitor molecule.
  • the CR2 -inhibitor molecule is purified by ion change chromatography.
  • the peptide may or may not be properly folded when expressed as a fusion protein. These factors determine whether the fusion protein must be denatured and refolded, and if so, whether these procedures are employed before or after cleavage.
  • a chaotrope such as a guanidine HC1
  • a redox buffer containing, for example, reduced and oxidized dithiothreitol or glutathione at the appropriate ratios, pH, and temperature, such that the peptide is refolded to its native structure.
  • the CR2-inhibitor molecules described herein may also contain a tag (such as a cleavable tag) for purification.
  • a tag such as a cleavable tag
  • This tag can be fused to the C-terminus or N- terminus of the CR2 portion or the inhibitor portion, and can be used to facilitate protein purification.
  • the CR2 -inhibitor molecule could be synthesized de novo in whole or in part, using chemical methods well known in the art.
  • the component amino acid sequences can be synthesized by solid phase techniques, cleaved from the resin, and purified by preparative high performance liquid
  • composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing.
  • the CR2-inhibitor molecules can be assayed for their desired properties using in vitro or in vivo assays. For example, binding of CR2-inhibitor to CR2 ligand can be determined by surface plasmon resonance method.
  • surface plasmon resonance method By way of example, kinetic analysis of the interaction of the molecule with C3dg-biotin can be performed using surface plasmon resonance (SPR) measurements made on a BIAcore 3000 instrument (Biacore AB, Uppsala, Sweden).
  • SPR surface plasmon resonance
  • Human C3dg-biotin can be bound to the surface of BIAcore streptavidin sensor chips by injecting C3dg-biotin over the surface of one flow cell of the chip.
  • Binding can be evaluated over a range of CR2-inhibitor molecule concentrations. Association of molecule with the ligand can be monitored for a certain period of time (such as 120 seconds), after which the complex is allowed to dissociate in the presence of buffer only for an additional period of time (such as 120 seconds).
  • Binding of CR2 fusion protein fragments to C3dg-immobilized flow cells can be corrected for binding to control flow cells.
  • Binding data can be fitted to a 1 : 1 Langmuir binding model using BIAevaluation Version 3.1 software (BIAcore) and evaluated for best fit.
  • the kinetic dissociation profiles obtained can be used to calculate on and off rates (ka and kd) and affinity constants (KD) using the BIAevaluation Version 3.1 program.
  • Other assay methods for ligand binding are known in the art and can also be used.
  • In vitro zymosan complement assay can be used to determine complement inhibitory activity of CR2-inhibitor molecules. Lysis of rabbit erythrocytes by serum in Mg-EGTA is another measure of activity that can be used. Lysis in Mg-EGTA of human or sheep erythrocytes that have had sialic acid removed provides for additional measures of activity.
  • the present invention relates to a composition
  • a composition comprising an immunosuppressant agent in combination with a CR2-inhibitor molecule.
  • the composition comprises a sub-therapeutic amount of an immunosuppressant agent in combination with a CR2-inhibitor molecule.
  • the immunosuppressant agent in combination with a CR2-inhibitor molecule.
  • the immunosuppressant agent is selected from a list comprising but not limited to cyclosporine A, azathioprine, corticosteroids including prednisone, and methylprednisolone, cyclophosphamide, FK506 and mTOR inhibitors including rapamycin, sirolimus, and everolimus.
  • the immunosuppressant agent is cyclosporine A which is an immunosuppressant drug widely used in organ transplantation to
  • the immunosuppressant agent is azathioprine, which is an immunosuppressive drug used in organ transplantation and autoimmune diseases and belongs to the chemical class of purine analogues.
  • Azathioprine is an imidazolyl derivative and prodrug of mercaptopurine.
  • the metabolites of mercaptopurine are incorporated into replicating DNA, halting replication, as well as blocking the pathway for purine synthesis. It thus most strongly affects proliferating cells, such as the T cells and B cells of the immune system.
  • the immunosuppressant agent is prednisone which is a synthetic corticosteroid drug that is used to treat certain inflammatory
  • the immunosuppressant agent is methylprednisolone which is a synthetic glucocorticoid or corticosteroid drug. It is a variant of prednisolone, methylated at carbon 6 of the B ring.
  • the immunosuppressant agent is cyclophosphamide which is an alkylating agent of the nitrogen mustard type (specifically, the
  • Cyclophosphamide is used to treat cancers, autoimmune disorders and AL amyloidosis. As a prodrug, it is converted by liver cytochrome P450 (CYP) enzymes to form the metabolite 4-hydroxy cyclophosphamide that has
  • the immunosuppressant agent is FK506 is an immunosuppressive drug used mainly after allogeneic organ transplant to lower the risk of organ rejection. It achieves this by inhibiting the production of interleukin-2, a molecule that promotes the development and proliferation of T cells.
  • the immunosuppressant agent is an inhibitor of mTOR selected from a group comprising rapamycin, sirolimus, and everolimus.
  • the present invention provides methods for enhancing survival of transplant tissue.
  • the method comprises administering a composition comprising a CR2-inhibitor molecule to a subject who is the recipient of a transplant tissue.
  • the method comprises administering a composition
  • the method comprises administering a composition comprising a CR2-inhibitor molecule to a subject before the subject has received the transplant tissue.
  • the method comprises administering a composition comprising a CR2-inhibitor molecule to a subject after the subject has received the transplant tissue.
  • the method comprises administering a composition comprising a CR2 -inhibitor molecule to transplant tissue ex vivo, prior to introducing the transplant tissue into a recipient subject.
  • the method comprises administering a composition comprising a CR2 -inhibitor molecule to a donor subject, prior to harvesting of the transplant tissue.
  • the method comprises administering a composition comprising a CR2 -inhibitor molecule in combination with an immunosuppressant agent to a subject or tissue. In one embodiment, the method comprises administering a composition comprising a CR2-inhibitor molecule in combination with a sub-therapeutic level of an immunosuppressant agent to a subject or tissue. In one embodiment, the immunosuppressant agent is selected from a list comprising but not limited to
  • cyclosporine A azathioprine
  • corticosteroids including prednisone
  • methylprednisolone cyclophosphamide
  • FK506 cyclophosphamide
  • mTOR inhibitors including rapamycin, sirolimus, and everolimus.
  • the composition comprises an amount of the immunosuppressant agent that is less than the amount necessary when the
  • the amount or concentration of immunosuppressant agent, when administered in combination with a targeted complement inhibitor described herein, is about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the amount or
  • the transplant graft is obtained and modified ex vivo, using the CR2-inhibitor and immunosuppressant agent composition described herein.
  • the transplant graft is obtained from a recipient, treated with the composition, and is then placed in contact or in the vicinity of the recipient.
  • the transplant graft is placed in a container comprising a suitable media and a composition comprising CR2-inhibitor molecules and immunosuppressant agent, or variant thereof.
  • the transplant graft is perfused with a suitable media and a composition comprising CR2 -inhibitor molecules and immunosuppressant agent, or variant thereof.
  • the composition comprising CR2 -inhibitor and sub-therapeutic immunosuppressant agent or variant thereof is administered to the transplant graft following removal or harvest of the graft from a donor.
  • the composition is administered to the transplant graft less than 72 hours, 48 hours, 36 hours, 24 hours, 18 hours, 12 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 45 minutes, 30 minutes, 15 minutes, 10 minutes, 5 minutes, or 1 minute following removal or harvest of the graft from a donor.
  • the composition is administered to the transplant graft for more than 72 hours, 48 hours, 36 hours, 24 hours, 18 hours, 12 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 45 minutes, 30 minutes, 15 minutes, 10 minutes, 5 minutes, or 1 minute prior to transplantation of the transplant graft into the recipient.
  • the CR2-inhibitor molecules described herein can function to specifically inhibit in vivo complement activation in the alternative complement pathway and inflammatory manifestations that accompany it, such as recruitment and activation of macrophages, neutrophils, platelets, and mast cells, edema, tissue damage, and direct activation of local and endogenous cells.
  • Compositions comprising these molecules can therefore be used for treatment of diseases or conditions that are mediated by excessive or uncontrolled activation of the complement system, particularly diseases or conditions mediated by excessive or uncontrolled activation of the alternative complement pathway.
  • methods of treating diseases associated with FH deficiencies for example a decrease in FH level, decrease in FH activity, or lack of wild type or protective FH
  • FH FH deficiencies
  • a method of treating a disease in which the alternative complement pathway is implicated in an individual comprising administering to the individual an effective amount of a composition comprising a CR2- inhibitor molecule comprising: a) a CR2 portion comprising a CR2 or a fragment thereof, and b) an inhibitor portion comprising an inhibitor (for example a FH molecule) or a fragment thereof.
  • a method of inhibiting complement activation in an individual having a disease in which the alternative complement pathway is implicated comprising administering to the individual an effective amount of a composition comprising a CR2 -inhibitor molecule comprising: a) a CR2 portion comprising a CR2 or a fragment thereof, and b) an inhibitor portion comprising an inhibitor molecule or a fragment thereof.
  • a method of inhibiting inflammation in an individual having a disease in which the alternative pathway is implicated comprising administering to the individual an effective amount of a composition comprising a CR2 -inhibitor molecule comprising: a) a CR2 portion comprising a CR2 or a fragment thereof, and b) an inhibitor portion comprising an inhibitor or a fragment thereof.
  • the disease to be treated is ischemia reperfusion.
  • Ischemia reperfusion (I/R) injury refers to inflammatory injury to the endothelium and underlying parenchymal tissues following reperfusion of hypoxic tissues. Ischemia reperfusion injury can result in necrosis and irreversible cell injury.
  • the complement pathway (including the alternative complement pathway) is a major mediator of I/R injury. Methods provided herein are thus useful for treatment of ischemia reperfusion that occurs in any organ or tissues, such as ischemia-reperfusion injury of any transplanted organ or tissue. Other conditions and diseases in which ischemia-reperfusion injury occurs will be known to those of skill in the art.
  • the present invention provides, methods of treating and/or preventing graft rejection with provided compositions.
  • graft rejection may be autograft rejection, xenograft rejection, and/or allograft rejection.
  • the present invention provides methods of treating and/or preventing graft-versus-host disease.
  • organ transplantation For dysfunctional, diseased, and/or otherwise undesired tissues or organs of the body, besides therapeutic intervention with drugs, organ and/or tissue transplantation is an alternative, and, in some cases, the last resort in the treatment of a patient. Particularly for patients with leukemia, end-stage renal, cardiac, pulmonary or hepatic failure, organ transplantation is quite commonly used in the treatment.
  • allografts organ grafts harvested from donors other than the patient him/herself or host/recipient of the graft
  • allografts of various types, e.g. kidney, heart, lung, liver, bone marrow, pancreas, cornea, small intestine and skin (e.g. epidermal sheets)
  • xenografts organ grafts harvested from non-human animals, such as porcine heart valves, may be used to replace their dysfunctional human counterparts.
  • tissue transplantation is or comprises bone marrow and/or stem cell transplantation.
  • Bone marrow and/or stem cell transplantation has applications in a wide variety of clinical settings, including solid organ transplantation.
  • a major goal in solid organ transplantation is the engraftment of the donor organ without a graft rejection immune response generated by the recipient, while preserving the immune-competence of the recipient against other foreign antigens.
  • nonspecific immunosuppressive agents such as cyclosporin A, azathioprine, corticosteroids including prednisone, and methylprednisolone, cyclophosphamide, and FK506 are used.
  • nonspecific immunosuppressive agents function by suppressing all aspects of the immune response, thereby greatly increasing a recipient's susceptibility to infections and diseases, including cancer. Accordingly, using the herein defined composition comprising a complement-targeted inhibitor in combination with a subtherapeutic dose of an immunotherapeutic agent, and more safe and efficacious treatment plan is made herein available.
  • tissue transplantation is or comprises hematopoietic tissue transplantation (e.g. bone marrow transplantation).
  • hematopoietic tissue transplantation e.g. bone marrow transplantation
  • a goal of hematopoietic tissue transplantation is to achieve the successful engraftment of donor cells within a recipient host, such that immune and/or hematopoietic chimerism results.
  • Chimerism is the reconstitution of the various compartments of the recipient's hematoimmune system with donor cell populations bearing MHC molecules derived from both, the allogeneic or xenogeneic donor, and a cell population derived from the recipient or, alternatively, the recipient's hematoimmune system compartments which can be reconstituted with a cell population bearing MHC molecules derived from only the allogeneic or xenogeneic marrow donor.
  • Chimerism may vary from 100% (total replacement by allogenic or xenogeneic cells) to low levels detectable only by molecular methods. Chimerism levels may vary over time and be permanent or temporary.
  • graft rejection refers to an autograft rejection, wherein the donor individual and recipient individual are the same (i.e., a patient's own tissue).
  • the present invention provides, among other things, methods of administering to a recipient organism who has received or will receive a transplant of one or more heterologous tissue components from a donor organism, a composition comprising encapsulated recipient organism tissue components.
  • one or more recipient tissue components are encapsulated within a nanoparticle. In some embodiments, one or more recipient tissue components are encapsulated within a microbial cell. In some embodiments, one or more provided compositions are administered prior to the transplant. In some embodiments, one or more provided compositions are administered subsequent to the transplant.
  • graft rejection refers to a xenograft rejection, wherein the donor and recipient are of different species.
  • xenograft rejection occurs when the donor species tissue carries a xenoantigen against which the recipient species immune system mounts a rejection response.
  • the present invention provides, among other things, methods of administering to a recipient organism who has received or will receive a transplant of one or more heterologous tissue components from a donor organism of different species a composition comprising encapsulated donor organism tissue components.
  • a composition comprising encapsulated donor organism tissue components.
  • donor porcine tissue components are isolated, encapsulated within nanoparticles, and administered to a recipient organism. Allograft Rejection
  • graft rejection refers to an allograft rejection, wherein the donor individual and recipient individual are of the same species.
  • allograft rejection occurs when the donor tissue carries an alloantigen against which the recipient immune system mounts a rejection response.
  • the present invention provides, among other things, methods of administering to a recipient organism who has received or will receive a transplant of one or more heterologous tissue components from a donor organism a composition comprising encapsulated donor organism tissue components.
  • one or more heterologous tissue components are encapsulated within a nanoparticle. In some embodiments, one or more heterologous tissue components are encapsulated within a microbial cell. In some embodiments, one or more heterologous tissue components are from a different species. In some embodiments, one or more provided compositions are administered prior to the transplant. In some embodiments, one or more provided compositions are administered subsequent to the transplant.
  • provided formulations may be used to treat patients with various forms of GvHD including acute and chronic GvHD that is either naive or refractory to conventional immunosuppressive agents such as steroids and cyclosporine A.
  • provided formulations may be used as prophylaxis to prevent onset of GvHD by pretreating (i.e., tolerizing) the transplant recipient (i.e. host) prior to the transplantation and/or treating the recipient (i.e., host) within a certain time window post transplantation.
  • provided formulations may be used to treat patients with various forms of GvHD.
  • the host e.g., transplant recipient
  • the host is treated with one or more compositions comprises herein after receiving one or more tissue transplantation (i.e., tissue graft, organ transplant, etc.).
  • tissue transplantation i.e., tissue graft, organ transplant, etc.
  • compositions described herein can be administered to an individual via any route, including, but not limited to, intravenous (e.g., by infusion pumps), intraperitoneal, intraocular, intra-arterial, intrapulmonary, oral, inhalation, intravesicular, intramuscular, intra-tracheal, subcutaneous, intrathecal, transdermal, transpleural, topical, inhalational (e.g., as mists of sprays), mucosal (such as via nasal mucosa),
  • compositions are administered systemically (for example by intravenous injection). In some embodiments, the compositions are administered locally (for example by intraarterial or intraocular injection). In some embodiments, the compositions are administered by ex vivo incubation or perfusion.
  • provided pharmaceutical formulations are administered to a subject in combination with one or more other therapeutic agents or modalities, for example, useful in the treatment of one or more diseases, disorders, or conditions treated by the relevant provided pharmaceutical formulation, so the subject is simultaneously exposed to both.
  • a provided tissue component composition is utilized in a pharmaceutical formulation that is separate from and distinct from the pharmaceutical formulation containing the other therapeutic agent.
  • a provided tissue component composition is admixed with the composition comprising the other therapeutic agent.
  • a provided tissue component composition is produced individually, and the provided tissue component composition is simply mixed with another composition comprising another therapeutic agent.
  • provided formulations can be administered concurrently with, prior to, or subsequent to, one or more other therapeutic agents (e.g., desired known
  • the therapies employed may achieve a desired effect for the same disorder (for example, a provided tissue component composition useful for treating transplant rejection may be administered concurrently with a known immunosuppressant therapeutic that is also useful for treating transplant rejection), or they may achieve different effects (for example, a provided tissue component composition that is useful for treating transplant rejection may be
  • provided tissue component compositions in accordance with the invention are administered with a second therapeutic agent.
  • the terms “in combination with” and “in conjunction with” mean that the provided CR2-inhibitor formulation can be administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics such as an
  • immunosuppressant agent including but not limited to a sub-therapeutic dose of such an immunosuppressant agent.
  • each substance will be administered at a dose and/or on a time schedule determined for that agent.
  • the method comprises administering a
  • composition comprising a combination of an immunosuppressive agent and a CR2- inhibitor described herein.
  • the method comprises administering one or more compositions.
  • the method comprises administering a first composition comprising an immunosuppressive agent and a second composition comprising a CR2 -inhibitor described herein.
  • the different compositions may be administered to the subject in any order and in any suitable interval.
  • the one or more compositions are administered simultaneously or near simultaneously.
  • the method comprises a staggered administration of the one or more compositions, where a first composition is
  • the method has an additive effect, wherein the overall effect of the administering a combination of therapeutic agents or procedures is approximately equal to the sum of the effects of administering each therapeutic agent or procedure alone. In other embodiments, the method has a synergistic effect, wherein the overall effect of administering a combination of therapeutic agents or procedures is greater than the sum of the effects of administering each therapeutic agent or procedure alone.
  • the host receives medications, chemotherapy (i.e., destroy host bone marrow), total body irradiation, and other antibody medications before receiving donor tissue transplant.
  • chemotherapy i.e., destroy host bone marrow
  • total body irradiation i.e., total body irradiation
  • other antibody medications before receiving donor tissue transplant.
  • chance of graft rejection is related to the match between the donor and host MHC antigens, the overall genetic relationship between donor and host, and the type of disease for which the transplantation has been performed.
  • immunosuppressive therapy includes administration of corticosteroids (e.g., prednisolone, hydrocortisone, etc.), calcineurin inhibitors (e.g., cyclosporine, tacrolimus, etc.), anti-proliferatives (e.g., azathioprine, mycophenolic acid, etc.), mTOR inhibitors (e.g., sirolimus, rapamycin, everolimus, etc.), and/or combinations thereof.
  • corticosteroids e.g., prednisolone, hydrocortisone, etc.
  • calcineurin inhibitors e.g., cyclosporine, tacrolimus, etc.
  • anti-proliferatives e.g., azathioprine, mycophenolic acid, etc.
  • mTOR inhibitors e.g., sirolimus, rapamycin, everolimus, etc.
  • corticosteroids are coadministered with one or more calcineurin inhibitors and one or more anti-proliferative agents.
  • mTOR inhibitors are used where calcineurin inhibitors or steroids are contraindicated.
  • antibody-based therapy includes administration of one or more antibodies or antibody -based drugs specific to select immune system components (e.g., IL-2R.0C. receptor, CD20, T-cells, etc.).
  • Non-limiting examples of antibodies or antibody-based drugs include monoclonal anti-IL-2R. a.
  • GvHD graft-versus-host disease
  • tissue transplant e.g. tissue implantation, graft, etc.
  • GvHD occurs when transplanted donor immune cells (e.g., white blood cells including T cells) present in the graft (i.e. donor) tissue recognize the host (i.e., recipient) as "non-self (i.e., antigenically "foreign") and attack the tissues of the recipient.
  • Donor infection-fighting cells attack tissues in the host just as if they were attacking an infection.
  • bone marrow transplant presents risk of GvHD, because mature donor lymphocytes present within transplanted donor marrow may recognize recipient tissues as foreign.
  • the transplanted donor immune cells then attack the host's body cells and destroy them.
  • GvHD is most commonly associated with stem cell or bone marrow transplant, but applies to other forms of tissue graft as well.
  • three criteria must be met in order for GvHD to occur.
  • an immune-competent graft i.e., donor tissue
  • viable and functional donor immune cells i.e., transplanted to host
  • the host is immunologically disparate (i.e., histo-incompatible).
  • the host is immune- compromised and cannot destroy or inactivate the transplanted donor immune cells.
  • lymphocytes i.e., T cells
  • TNF-alpha TNF-alpha
  • IFNy interferon-gamma
  • MHC- identical siblings or MHC-identical unrelated donors often have genetically different proteins (i.e., minor histocompatibility antigens) that can be presented by MHC molecules to the donor's T-cells, which see these antigens as foreign and so mount an immune response.
  • minor histocompatibility antigens i.e., minor histocompatibility antigens
  • donor T-cells are undesirable as effector cells of GvHD, they are valuable for engraftment by preventing the host residual immune system from rejecting the bone marrow graft (i.e., host-versus-graft; discussed above).
  • GvHD is either acute GvHD (aGVHD) or chronic
  • GvHD cGVHD
  • Acute GvHD usually occurs within the first three months following a transplant, and can affect the skin, liver, stomach, and/or intestines.
  • symptoms of aGvHD include rash, yellow skin and eyes due to elevated concentrations of bilirubin, and diarrhea.
  • Acute GvHD is graded on a scale of 1 to 4; grade 4 is the most severe. In some embodiments, aGvHD can be fatal.
  • Chronic GvHD (cGvHD) is the late form of the disease, and usually develops three months or more after a transplant.
  • the symptoms of cGvHD resemble spontaneously occurring autoimmune disorders such as lupus or scleroderma.
  • GvHD is more easily prevented than treated.
  • Preventive measures include the administration of cyclosporin with or without methotrexate or steroids after stem cell transplant.
  • T lymphocytes are removed from the stem cell graft before it is transplanted.
  • first-line treatment of GvHD includes steroid therapy.
  • chronic GvHD occurs approximately in 10-40 percent of patients after stem cell transplant.
  • symptoms vary more widely than those of acute GvHD and are similar to various autoimmune disorders.
  • symptoms include dry eyes, dry mouth, rash, ulcers of the skin and mouth, joint contractures (i.e., inability to move joints easily), abnormal blood test results of liver function, stiffening of the lungs (i.e., difficulty in breathing), inflammation in the eyes, difficulty in swallowing, muscle weakness, a white film in the mouth, and/or combinations thereof.
  • the incidence of GvHD increases with increasing degree of mismatch between donor and host HLA antigens, increasing donor age and increasing host age.
  • compositions comprising a CR2- inhibitor molecule and a pharmaceutically acceptable carrier.
  • the pharmaceutical compositions may be suitable for a variety of modes of administration described herein, including for example systemic or localized administration.
  • the pharmaceutical compositions can be in the form of eye drops, injectable solutions, or in a form suitable for inhalation (either through the mouth or the nose) or oral administration.
  • the pharmaceutical compositions described herein can be packaged in single unit dosages or in multidosage forms.
  • the pharmaceutical compositions comprise a pharmaceutically acceptable carrier suitable for administration to human. In some embodiments, the pharmaceutical compositions comprise a pharmaceutically acceptable carrier suitable for intraocular injection. In some embodiments, the pharmaceutical compositions comprise a pharmaceutically acceptable carrier suitable for topical application. In some embodiments, the pharmaceutical compositions comprise a pharmaceutically acceptable carrier suitable for intravenous injection. In some embodiments, the pharmaceutical compositions comprise and a pharmaceutically acceptable carrier suitable for injection into the arteries.
  • compositions are generally formulated as sterile, substantially isotonic, and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.
  • GMP Good Manufacturing Practice
  • the composition is free of pathogen.
  • the pharmaceutical composition can be in the form of liquid solutions, for example in physiologically compatible buffers such as Hank's solution or Ringer's solution.
  • the CR2- inhibitor pharmaceutical composition can be in a solid form and redissolved or suspended immediately prior to use. Lyophilized compositions are also included.
  • the pharmaceutical compositions can take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate).
  • binding agents e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose
  • fillers e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate
  • lubricants e.g., magnesium stearate, talc or silica
  • disintegrants e.g., potato star
  • Liquid preparations for oral administration can take the form of, for example, solutions, syrups or suspensions, or they can be presented as a dry product for constitution with water or other suitable vehicle before use.
  • Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., ationd oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid).
  • suspending agents e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats
  • emulsifying agents e.g., lecithin or acacia
  • non-aqueous vehicles e.g., ationd oil, oily esters,
  • preparations can also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.
  • compositions comprising a CR2-inhibitor molecule and a pharmaceutically acceptable carrier suitable for administration to the eye.
  • pharmaceutical carriers can be sterile liquids, such as water and oil, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, and the like.
  • Saline solutions and aqueous dextrose, polyethylene glycol (PEG) and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, sodium state, glycerol monostearate, glycerol, propylene, water, and the like.
  • the pharmaceutical composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • the CR2 -inhibitor molecule and other components of the composition may be encased in polymers or fibrin glues to provide controlled release of the molecule.
  • compositions can take the form of solutions, suspensions, emulsions, ointment, gel, or other solid or semisolid compositions, and the like.
  • the compositions typically have a pH in the range of 4.5 to 8.0.
  • the compositions must also be formulated to have osmotic values that are compatible with the aqueous humor of the eye and ophthalmic tissues. Such osmotic values will generally be in the range of from about 200 to about 400 milliosmoles per kilogram of water (“mOsm/kg”), but will preferably be about 300 mOsm/kg.
  • the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for injection intravenously, intraperitoneally, or intravitreally.
  • compositions for injection are solutions in sterile isotonic aqueous buffer.
  • the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
  • compositions may further comprise additional ingredients, for example preservatives, buffers, tonicity agents, antioxidants and stabilizers, nonionic wetting or clarifying agents, viscosity-increasing agents, and the like.
  • additional ingredients for example preservatives, buffers, tonicity agents, antioxidants and stabilizers, nonionic wetting or clarifying agents, viscosity-increasing agents, and the like.
  • Suitable preservatives for use in a solution include polyquaternium-1, benzalkonium chloride, thimerosal, chlorobutanol, methyl paraben, propyl paraben, phenylethyl alcohol, edetate disodium, sorbic acid, benzethonium chloride, and the like.
  • such preservatives are employed at a level of from 0.001% to 1.0% by weight.
  • Suitable buffers include boric acid, sodium and potassium bicarbonate, sodium and potassium borates, sodium and potassium carbonate, sodium acetate, sodium biphosphate and the like, in amounts sufficient to maintain the pH at between about pH 6 and pH 8, and preferably, between about pH 7 and pH 7.5.
  • Suitable tonicity agents are dextran 40, dextran 70, dextrose, glycerin, potassium chloride, propylene glycol, sodium chloride, and the like, such that the sodium chloride equivalent of the ophthalmic solution is in the range 0.9 plus or minus 0.2%.
  • Suitable antioxidants and stabilizers include sodium bisulfite, sodium metabi sulfite, sodium thiosulfite, thiourea and the like.
  • Suitable wetting and clarifying agents include polysorbate 80, polysorbate 20, poloxamer 282 and tyloxapol.
  • Suitable viscosity-increasing agents include dextran 40, dextran 70, gelatin, glycerin,
  • hydroxyethylcellulose hydroxmethylpropylcellulose, lanolin, methylcellulose, petrolatum, polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone,
  • viscosity enhancing agents to provide topical compositions with viscosities greater than the viscosity of simple aqueous solutions may be desirable.
  • viscosity building agents include, for example, polyvinyl alcohol, polyvinyl pyrrolidone, methyl cellulose, hydroxy propyl methylcellulose, hydroxyethyl cellulose, carboxymethyl cellulose, hydroxy propyl cellulose or other agents know to those skilled in the art. Such agents are typically employed at a level of from 0.01% to 2% by weight.
  • a pharmaceutical composition for delivery of a nucleotide encoding a CR2-inhibitor molecule can be in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle or compound is imbedded.
  • the pharmaceutical composition can comprise one or more cells which produce the gene delivery system.
  • a gene delivery system for a gene therapeutic can be introduced into a subject by any of a number of methods.
  • a pharmaceutical composition of the gene delivery system can be introduced systemically, e.g., by intravenous injection, and specific transduction of the protein in the target cells occurs predominantly from specificity of transfection provided by the gene delivery vehicle, cell-type or tissue-type expression due to the transcriptional regulatory sequences controlling expression of the receptor gene, or a combination thereof.
  • initial delivery of the recombinant gene is more limited with introduction into the animal being quite localized.
  • the gene delivery vehicle can be introduced by catheter, See U.S. Pat. No. 5,328,470, or by stereotactic injection, Chen et al. (1994), Proc.
  • a polynucleotide encoding a CR2 -inhibitor molecule can be delivered in a gene therapy construct by electroporation using techniques described, Dev et al. (1994), Cancer Treat. Rev. 20: 105-115.
  • compositions can be determined empirically and will depend on the type and severity of the disease, route of administration, and
  • the effective amount can also be determined based on in vitro complement activation assays.
  • dosages of CR2 -inhibitor molecules which can be used for methods described herein include, but are not limited to, an effective amount within the dosage range of any of about 0.01 mg/kg to about 300 mg/kg, or within about 0.1 mg/kg to about 40 mg/kg, or with about 1 mg/kg to about 20 mg/kg, or within about 1 mg/kg to about 10 mg/kg.
  • the amount of CR2-FH administered to an individual is about 10 mg to about 500 mg per dose, including for example any of about 10 mg to about 50 mg, about 50 mg to about 100 mg, about 100 mg to about 200 mg, about 200 mg to about 300 mg, about 300 mg to about 500 mg, about 500 mg to about 1 mg, about 1 mg to about 10 mg, about 10 mg to about 50 mg, about 50 mg to about 100 mg, about 100 mg to about 200 mg, about 200 mg to about 300 mg, about 300 mg to about 400 mg, or about 400 mg to about 500 mg per dose.
  • the CR2-inhibitor compositions may be administered in a single daily dose, or the total daily dose may be administered in divided dosages of two, three, or four times daily.
  • the compositions can also be administered less frequently than daily, for example, six times a week, five times a week, four times a week, three times a week, twice a week, once a week, once every two weeks, once every three weeks, once a month, once every two months, once every three months, or once every six months.
  • the compositions may also be administered in a sustained release formulation, such as in an implant which gradually releases the composition for use over a period of time, and which allows for the composition to be administered less frequently, such as once a month, once every 2-6 months, once every year, or even a single administration.
  • the sustained release devices (such as pellets, nanoparticles, microparticles, nanospheres, microspheres, and the like) may be administered by injection or surgical implantation in various locations.
  • Dosage amounts and frequency will vary according the particular formulation, the dosage form, and individual patient characteristics. Generally speaking, determining the dosage amount and frequency for a particular formulation, dosage form, and individual patient characteristic can be accomplished using conventional dosing studies, coupled with appropriate diagnostics.
  • unit dosage forms of CR2- inhibitor molecule compositions each dosage containing from about 0.01 mg to about 50 mg, including for example any of about 0.1 mg to about 50 mg, about 1 mg to about 50 mg, about 5 mg to about 40 mg, about 10 mg to about 20 mg, or about 15 mg of the CR2-inhibitor molecule.
  • the unit dosage forms of CR2- inhibitor molecule composition comprise about any of 0.01 mg-0.1 mg, 0.1 mg-0.2 mg, 0.2 mg-0.25 mg, 0.25 mg-0.3 mg, 0.3 mg-0.35 mg, 0.35 mg-0.4 mg, 0.4 mg-0.5 mg, 0.5 mg-1.0 mg, 10 mg-20 mg, 20 mg- 50 mg, 50 mg-80 mg, 80 mg-100 mg, 100 mg-150 mg, 150 mg-200 mg, 200 mg-250 mg, 250 mg-300 mg, 300 mg-400 mg, or 400 mg-500 mg CR2- inhibitor molecule.
  • the unit dosage form comprises about 0.25 mg CH2- inhibitor molecule.
  • unit dosage form refers to a physically discrete unit suitable as unitary dosages for an individual, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical carrier, diluent, or excipient.
  • suitable pharmaceutical carrier diluent, or excipient.
  • unit dosage forms can be stored in suitable packaging in single or multiple unit dosages and may also be further sterilized and sealed.
  • kits comprising compositions (or unit dosages forms and/or articles of manufacture) described herein and may further comprise instruction(s) on methods of using the composition, such as uses described herein.
  • the kits described herein may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for performing any methods described herein.
  • Example 1 Targeted complement inhibition protects vascularized composite allografts from acute graft injury and prolongs graft survival when combined with subtherapeutic cyclosporine A therapy.
  • mice Male Balb/c (H-2 d ) and C57BL/6 (B6; H-2 b ) mice (10-12 weeks) were obtained from Jackson laboratory (Bar Harbor, ME). Male CSaR ⁇ ' CSaR " " and C3-/- C57BL/6 mice were obtained. All mice weighed 25-30 g and were housed under specific pathogen-free conditions.
  • mice were used as donors, and C57B1/6, CSaR ⁇ ' CSaR " " and C3 _/"
  • mice were used as recipients.
  • a heterotopic hindlimb Tx model was used.
  • animals were randomized into four groups: 1. Balb/c donors and wild type (WT) B6 recipients with PBS treatment, 2. Balb/c donors and CSaR ⁇ ' CSaR " " B6 recipients, 3. Balb/c donors and C3 _/" B6 recipients, 4. Balb/c donors and WT B6 recipients administered two doses of 0.25 mg CR2-Crry complement inhibitor immediately upon completion of surgery, and again 24 hours posttransplantation.
  • WT wild type
  • mice were anesthetized with intraperitoneal injections of ketamine (75 mg/kg) and xylazine (16 mg/kg) diluted in sterile saline, and the cold ischemia time for all grafts was 2 h.
  • CR2-Crry was produced and purified as previously described (Atkinson et al., 2005, The Journal of clinical investigation, 115(9): 2444-2453). Dose was based on previously published data (Atkinson et al., 2005, The Journal of clinical investigation, 115(9): 2444-2453, Huang et al., 2008, Journal of immunology, 181(11): 8068-8076). Histopathology
  • VCAs were isolated and subsequently placed in 10% buffered formaldehyde solution for 48 hours before being embedded in paraffin.
  • Four ⁇ graft sections were stained with Hematoxylin and Eosin (HE) and scored using a semiquantitative histology scoring system as described (Hautz et al., 2014, Transplantation, 98(7): 713-720).
  • HE Hematoxylin and Eosin
  • Splenocytes were isolated from naive mice, and from mice that had received Balb/c allografts from four groups; 1. B6 recipient, no treatment, 2. B6 recipient, treatment with 3mg/kg/day CsA, 3. B6 recipient, treatment with CR2-Crry, and 4. B6 recipient, treatment with CR2-Crry + 3mg/kg/day CsA. Spleens were isolated 7 days post-transplant and splenocytes isolated and frozen for later analysis.
  • splenocytes were stained for cell surface markers (CD3 eFluor450, CXCR3 FITC; eBioscience; CD4 APC Cy7, CD8 APC, CD44 PerCP Cy5.5, CD62L FITC, FoxP3 PE, BD Biosciences) in FACS buffer (PBS + 2% FBS) for 20 minutes at room temperature. Cells were then washed and incubated in Fixation Buffer (BioLegend) for 10 minutes. After washing, cells were run on a BD Verse (BD Biosciences) and analyzed using Flow Jo software (Tree Star, Ashland, OR).
  • GraphPad Prism version 5.0 for Mac OS X (GraphPad, San Diego, CA) was used for statistical analysis. A student t test was used for the comparison of a normally distributed continuous variable between two groups. Differences between various groups were compared by the nonparametric Wilcoxon rank-sum or Mann- Whitney test because of the small sample sizes for some experiments. The Mantel Cox text was used to compare survival curves for different groups on univariate analyses. All analyses were 2 sided, and values of P ⁇ 0.05 were considered to be statistically significant.
  • Vascularized composite allografts from Balb/c WT donors were transplanted into the following C57BL/6 recipients: 1. WT mice treated with PBS vehicle at 0 and 24 h post-reperfusion; 2. Double CSaR ⁇ VCSaR " " mice; 3. C3 _/” mice; 4. WT mice treated with 0.25 mg CR2-Crry at 0 and 24 hours post-reperfusion. Skin and muscle tissue from allografts was isolated 48 h after Tx and assessed for injury and cellular infiltration. Allografts isolated from PBS treated WT recipients exhibited key features associated with IRI, including epidermal and muscle cell necrosis, and inflammatory cell infiltration of the epidermis localized to areas of muscle necrosis (Figure 1).
  • organ IRI The paradigm for organ IRI is that following reperfusion, circulating self- reactive natural IgM Abs bind to ischemia-induced neoepitopes or danger associated molecular patterns (DAMPs) and activate complement (Ioannou et al., 2011, Clinical immunology, 141(1): 3-14). The binding of IgM and deposition of the C3d complement activation product in allografts was then investigated. At 48 h post-transplant, IgM staining was evident in both skin and muscle tissue, with a similar pattern and intensity in allografts from all recipient groups ( Figure 2 A and B).
  • C3d deposition was significantly lower in both the skin and muscle of allografts from C3 deficient and CR2-Crry treated recipients when compared to allografts from WT and CSaR ⁇ VCSaR " " mice (donor-derived complement can account for the low levels of C3d deposition observed in allografts from C3-/- recipients). Furthermore, IgM and C3d co-localized in allografts from WT mice, indicating IgM-mediated activation of complement ( Figure 2C). These data indicate sequential IgM binding and complement activation, since neither complement deficiency nor inhibition affected IgM binding. The data further
  • the anaphylatoxins in particular C5a, are known to play important roles in the recruitment of immune cells to sites of inflammation, allograft infiltration of neutrophils and macrophages 48 h after Txm, was investigated.
  • Immunohistological assessment demonstrated extensive infiltration of both cell types in allografts from control WT recipients. Neutrophils and macrophages were localized to the dermis in the skin and spread between myocytes in muscle tissue. Compared to WT recipients, the number of infiltrating cells was significantly reduced in allografts from CSaR ⁇ VCSaR " " recipients, with the exception of skin neutrophils.
  • the adhesion molecule P-selectin is expressed on endothelial cells early after IR, and it is an important mediator of leukocyte infiltration and injury, at least in some organs and tissues. Adhesion molecules also modulate the infiltration of lymphocytes involved in alloimmunity. Moreover, there is a dynamic between P-selectin expression and complement activation (Del Conde et al., 2005, The Journal of experimental medicine, 201(6): 871-879), and therefore the effect of complement deficiency and inhibition on P-selectin expression in VC allografts was investigated.
  • CsA Cyclosporine A
  • Vascularized composite allograft rejection is primarily dependent on T cells, and demonstrates that complement activation within donor organs contributes to post-transplant ischemic injury and memory T cell infiltration and proliferation. It was then determined how CR2-Crry treatment affected graft rejection and T cell infiltration into allografts when recipients were co-treated with a subtherapeutic 3 mg/kg/day dose of CsA. Graft rejection was evident macroscopically in untreated recipients and in recipients treated with a subtherapeutic dose of CsA alone at day 7 post-transplant (Figure 6A).
  • VCA surgical procedures have become highly successful, the high incidence of acute rejection within the first year after surgery (Fischer et al., 2014, Current opinion in organ transplantation, 19(6): 531-544) and the requirement for long- term and high-dose immunosuppression (Fischer et al., 2014, Current opinion in organ transplantation, 19(6): 531-544, Napoli et al., 2012, Neuron, 73(4): 729-742) remain obstacles to its widespread application.
  • Current immunosuppression strategies applied in VCA are extrapolated from solid organ Tx regimens (Petruzzo et al., 2010,
  • C3aR/C5aR deficiency in the recipient was protective against IRI
  • C3 deficiency and C3 inhibition provided significantly better protection in both skin and muscle compartments.
  • the anaphylatoxins may contribute to IRI, via the recruitment and/or activation of leukocytes, C3 opsonins and/or the MAC play a prominent role in VCA IRI.
  • the MAC appears to be the primary mediator of IRI (Zhang et al., 2011, The American journal of pathology, 179(6): 2876-2884, Turnberg et al., 2004, The American journal of pathology, 165(3): 825-832, Marshall et al., 2014, The Journal of experimental medicine, 211(9): 1793-1805, Austen et al., 1999, Surgery, 126(2): 343-348).
  • deposition of the MAC at sublytic concentrations can, as can the anaphylatoxins, result in the activation of endothelial cells and the recruitment and activation of leukocytes (Morgan BP et al., 2015, Immunobiology, 221(6): 747-751, Merle et al., 2015, Frontiers in immunology, 6: 257).
  • Activated endothelial cells and recruited macrophages function as APCs, and C3 opsonization can facilitate cell/antigen uptake via complement receptor engagement.
  • Complement-dependent injury will also result in the expression of DAMPs, such as heat shock proteins, reactive oxygen species and fibrinogen (Caterson et al., 2013, The Journal of craniofacial surgery.
  • the data also demonstrates IgM-mediated activation of complement occurs in composite allografts, and for cardiac grafts it has been demonstrated that DAMPs exposed after transplant are recognized by self-reactive natural IgM Abs that activate complement in the transplanted heart (Atkinson et al., 2015, Circulation, 131(13): 1171-1180).
  • the activities of complement activation products provide a basis for how complement may augment graft allogenicity via IRI (Caterson et al., 2013, The Journal of craniofacial surgery. 24(1): 51-56).
  • C3a and C5a receptors expressed on APCs and T cells can stimulate differentiation, expansion and survival of effector T cells, and C3a/C5a receptor signaling can also reduce the suppressive activity of regulatory T cells, thus promoting T cell alloimmunity (Kwan et al., 2012, Immunologic research, 54(1-3): 247-253).
  • complement activation products C3a, C5a, C3 opsonins, and the MAC have all been implicated in acute graft inflammation and injury following solid organ Tx, and organ IRI is implicated in promoting the development of graft rejection.
  • CR2-Crry is a pan-complement inhibitor that inhibits the generation of all of the above complement activation products, and the data shown here demonstrates that a treatment directed at inhibiting complement-mediated IRI of VC allografts is a promising therapeutic strategy.
  • CR2-mediated targeting of complement inhibition significantly increases bioavailability and efficacy, and obviates the need for systemic inhibition that reduces any additional immunosuppressive effect, a potentially important consideration when the patient is immune-compromised.
  • Example 2 Inhibition of Alternative Complement pathway in Target Organs represents a novel and effective approach to control GVHD while sparing GVL Effect
  • Allogeneic hematopoietic stem cell transplantation is an effective immunotherapy for hematological malignancies.
  • application of allo- HSCT is limited by graft-versus-host-disease (GVHD), and separation of GVHD and graft-versus-leukemia (GVL) is a great challenge in the field.
  • Complement can be activated via three different pathways: classical, lectin and alternative pathway. Owning to the auto-activation property and crucial in "amplification loop", alternative complement pathway (ACP) plays a central role in the development of autoimmune diseases such as colitis and inflammatory bowel disease, which share certain
  • CR2-fH consists of the N-terminus of mouse complement factor H, which contains the APC-inhibitory domain, linked to a complement receptor 2 (CR2) targeting fragment that binds complement activation product C3d which highly expressed at GVHD target organs ( Figure 13).
  • mice deficient for fB or Clq/MLB were used as recipients in which the ACP or classical/actin pathway is altered.
  • MHC-mismatched murine models of GVHD it was determined that GVHD severity and mortality was significantly reduced in fB but not Clq/MLB deficient recipients compared to wild type (WT) counterpart, suggesting a crucial role of ACP in GVHD pathogenesis after allo-HSCT, as shown in Figure 15 A. It has shown that CR2-fH co-localizes with C3d in injured issues.
  • donor T cells and particularly CD103+CD8+ T cells were decreased in the gut of fB-/- recipients. Consistently, in the fB-/- recipients, donor T cells reduced the expression of gut homing chemokine CCR9. Donor Thl and Tel cells were significantly decreased whereas donor iTregs were increased in the liver of fB-/- recipients ( Figure 16B). Meanwhile, donor Th2 and Tel were diminished in the lung. The cytolytic activity of liver-infiltrated lymphocyte was also lower in the absence of host ACP. The thymic integrity was significantly improved in fB-/- recipients. No differences in differentiation and function of donor T cells in lymphoid organs were found in the fB-/- recipients ( Figure 16C). These studies suggest that complement activation in target organs is important for GVHD pathogenesis.
  • this study identifies a central role of ACP in GVHD and validates ACP as a therapeutic target for the control of GVHD. It provides evidences that locally-generated complement at GVHD target organs is important for GVHD
  • Example 3 In vitro characterization of Map44-CR2.
  • a construct was designed which includes MAp44 linked to CR2 (MAp44- CR2; SEQ ID NO: 16).
  • a binding (targeting) assay is conducted where an ELISA plate coated with C3d or BSA is incubated with various concentrations of MAp44-C4. The plates are then washed and binding is detected by means of anti-Map44 mAb.
  • a lectin pathway inhibition assay is conducted using a lectin pathway-specific assay using mannan coated ELISA plates ( Figure 17)
  • Example 4 CR2-Crry and subtherapeutic rapamycin therapy.
  • rapamycin potent Treg inducing capacity is well documented it is seldom used in clinical Tx due to severe side-effects associated with poor wound healing, patient tolerability issues, infection and cancer.
  • a number of novel strategies have been employed in an attempt to utilize rapamycin, one such is the use of rapamycin at subtherapeutic doses combine with ex-vivo expanded Tregs to support therapeutic levels of Treg proliferation in vivo.
  • the strategy described herein employs a sub-therapeutic rapamycin dosing strategy, that does not alone protect against AV development, combined with early complement inhibition with CR2-Crry (C3 convertase inhibitor) and anti-C5 (C5 and MAC inhibitor) administered at 0 and 24 hrs post reperfusion.
  • CR2-Crry C3 convertase inhibitor
  • anti-C5 C5 and MAC inhibitor
  • Six groups are utilized, 1. No treatment control, 2. Rapamycin administered at 300ng dose on days 7, 9, 10, and 11 post transplantation, 3. CF2-Crry alone, 4. Anti-C5 alone, 5. B4-Crry + Rapamycin, and 6. Anti-C5 + Rapamycin.
  • CR2-Crry was added to the University of Wisconsin (UW) solution which was perfused through the donor. The vessels were clamped and the grafts were stored in UW solution for 6 hours. Living donors (LD) and brain dead (BD) donors were used in the experiments.
  • UW University of Wisconsin
  • IRI damage was assessed in the vascularized composite allografts from recipients of either living donor or brain dead donor grafts that had either been perfused with UW or UW augmented with CR2-Crry . Histological quantification of injury is shown in skin (Figure 19A) and muscle (Figure 19C) of grafts isolated at 48 hours posttransplant. Representative histological images is shown of skin ( Figure 19B) and muscle ( Figure 19D). It is demonstrated that treatment of BD donors with CR2-Crry significantly decreased skin and muscle histopathological score.
  • Figure 20 demonstrates that perfusion of grafts with UW solution augmented with CR2-Crry prolong graft survival of recipients of both living or brain dead donor grafts. There was no significant difference in graft survival in brain dead or living donors.

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Abstract

L'invention concerne des compositions et des procédés pour améliorer la survie d'un tissu greffé.
PCT/US2017/056898 2016-10-17 2017-10-17 Compositions et procédés de traitement et de prévention d'une lésion associée à une greffe WO2018075462A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109078168A (zh) * 2018-07-26 2018-12-25 广西医科大学第附属医院 靶向补体抑制剂在制备改善脑死亡供肝药物中的应用
WO2020041644A1 (fr) * 2018-08-22 2020-02-27 Alexion Pharmaceuticals, Inc. Peptides et méthodes de traitement d'affections liées à la dystrophie les utilisant
WO2021163472A1 (fr) * 2020-02-14 2021-08-19 The General Hospital Corporation Surveillance de la viabilité d'organes en vue d'une greffe
CN114532982A (zh) * 2022-01-12 2022-05-27 高谋 评价干细胞系统移植调控补体活化作用的体系

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160184391A1 (en) * 2013-08-16 2016-06-30 Alexion Pharmaceuticals, Inc. Treatment of graft rejection by administering a complement inhibitor to an organ prior to transplant

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160184391A1 (en) * 2013-08-16 2016-06-30 Alexion Pharmaceuticals, Inc. Treatment of graft rejection by administering a complement inhibitor to an organ prior to transplant

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109078168A (zh) * 2018-07-26 2018-12-25 广西医科大学第附属医院 靶向补体抑制剂在制备改善脑死亡供肝药物中的应用
CN109078168B (zh) * 2018-07-26 2019-09-06 广西医科大学第一附属医院 靶向补体抑制剂在制备改善脑死亡供肝药物中的应用
WO2020041644A1 (fr) * 2018-08-22 2020-02-27 Alexion Pharmaceuticals, Inc. Peptides et méthodes de traitement d'affections liées à la dystrophie les utilisant
WO2021163472A1 (fr) * 2020-02-14 2021-08-19 The General Hospital Corporation Surveillance de la viabilité d'organes en vue d'une greffe
CN114532982A (zh) * 2022-01-12 2022-05-27 高谋 评价干细胞系统移植调控补体活化作用的体系

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