# US 2018/0024140 A1 · Atomic description of immune complex that causes heparin-induced thrombocytopenia

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- Number: US 2018/0024140 A1
- Document ID: US20180024140A1
- Status: Published application
- Kind: A1
- Application number: 15/524,511
- Filed: 2015-11-05
- Published: 2018-01-25
- Earliest claimed: 2014-11-06
- Assignees: The Trustees of the University of Pennsylvania
- Applicants: The Trustees of The University of Pennsylvania
- Inventors: Mark I. GREENE; Douglas B. CINES; Zheng CAI; Zhiqlang ZHU
- Web page: https://patentgrep.com/patent/US20180024140A1

## CPC

- G01N 33/6863: {Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors}
- A61K 38/195: {Chemokines, e.g. RANTES}
- A61K 39/3955: {against proteinaceous materials, e.g. enzymes, hormones, lymphokines}
- C07K 14/522: {Alpha-chemokines, e.g. NAP-2, ENA-78, GRO-alpha/MGSA/NAP-3, GRO-beta/MIP-2alpha, GRO-gamma/MIP-2beta, IP-10, GCP-2, MIG, PBSF, PF-4, KC}
- C07K 16/24: against cytokines, lymphokines or interferons
- G01N 33/86: involving blood coagulating time {or factors, or their receptors}
- A61K 2039/505: {comprising antibodies}
- G01N 2333/522: Alpha-chemokines, e.g. NAP-2, ENA-78, GRO-alpha/MGSA/NAP-3, GRO-beta/MIP-2alpha, GRO-gamma/MIP-2beta, IP-10, GCP-2, MIG, PBSF, PF-4 or KC
- G01N 2400/40: Glycosaminoglycans, i.e. GAG or mucopolysaccharides, e.g. chondroitin sulfate, dermatan sulfate, hyaluronic acid, heparin, heparan sulfate, and related sulfated polysaccharides
- G01N 2800/222: Platelet disorders
- G01N 2800/50: Determining the risk of developing a disease
- G01N 2800/52: Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## Abstract

The present invention provides a humanized antibody or antibody fragment comprising (a) a humanized light chain comprising 1) Complementarity Determining Region (CDR)-L1, the sequence of which is identical to the sequence of SEQ ID NO: 3; 2) CDR-L2, the sequence of which is identical to the sequence of SEQ ID NO: 4; and 3) CDR-L3, the sequence of which is identical to the sequence of SEQ ID NO: 5, and (b) a humanized heavy chain comprising 1) CDR-H1, the sequence of which is identical to the sequence of SEQ ID NO: 6; 2) CDR-H2, the sequence of which is identical to the sequence of SEQ ID NO: 7; and 3) CDR-H3, the sequence of which is identical to the sequence of SEQ ID NO: 8, as well as methods for treating, diagnosing, and monitoring the progression of HIT. The present invention also provides methods for assessing the antigenicity and ability to cause HIT of anionic anticoagulants. The present invention also provides a mutant protein which has the same amino acid sequence of a wild type PF4 monomer except that (i) at least one amino acid of the wild type PF4 monomer has been deleted, (ii) at least one amino acid of the wild type PF4 monomer has been replaced by another amino acid, or (iii) a combination of such changes has been made. The present invention also provides methods of treating or reducing the likelihood of HIT, treating angiogenesis, treating abnormal cell growth, or affecting coagulation pathologies that lead to thrombus formation, by administering such mutant proteins to a patient.

## Claims

### Claim at position 1

1-15. (canceled)

### Claim 16

A method for detecting heparin-induced thrombocytopenia (HIT) in a subject, comprising
i) obtaining a plasma or serum sample from the subject; and
ii) detecting whether HIT is present by determining the ratio of
(1) the reactivity of antibodies in the sample to wild-type PF4, to
(2) the reactivity of antibodies in the sample to a mutant PF4 monomer that remains in a monomeric state.

### Claim 17 (depends on 16)

The method of claim 16, wherein the subject is diagnosed as having HIT if the ratio of (1) to (2) in the sample is equal to or greater than a reference ratio of (1) to (2) obtained from a plasma or serum sample from a subject or group of subjects afflicted with HIT.

### Claim at position 4

18-20. (canceled)

### Claim 21 (depends on 16)

The method of claim 16, wherein determining the ratio of (1) and (2) comprises ELISA.

### Claim 22 (depends on 21)

The method of claim 21, wherein the ratio of (1) to (2) is obtained using the optical density for (1) and the optical density for (2) obtained using ELISA.

### Claim at position 7

23-25. (canceled)

### Claim 26 (depends on 16)

The method of claim 16, wherein heparin is being or has been administered to the subject.

### Claim 27 (depends on 26)

The method of claim 26, wherein the heparin is low-molecular-weight heparin or unfractionated heparin.

### Claim 28 (depends on 16)

The method of claim 16, wherein a heparin mimic is being or has been administered to the subject.

### Claim 29 (depends on 28)

The method of claim 28, wherein the heparin mimic is fondaparinux.

### Claim at position 12

30-39. (canceled)

### Claim 40

A mutant platelet factor 4 (PF4) monomer which has the same amino acid sequence of wild type PF4 monomer except that
(i) at least one amino acid of the wild type PF4 monomer has been deleted,
(ii) at least one amino acid of the wild type PF4 monomer has been replaced by another amino acid, or
(iii) a combination of such changes has been made.

### Claim 41 (depends on 40)

The mutant platelet factor 4 (PF4) monomer of claim 40, wherein the at least one amino acid that has been deleted or replaced is on the N-terminal end of the wild type PF4 monomer.

### Claim 42 (depends on 40)

The mutant platelet factor 4 (PF4) monomer of claim 40, wherein at least 8 amino acids are deleted from the N-terminal end of a wild type PF4 monomer.

### Claim 43 (depends on 42)

The mutant platelet factor 4 (PF4) monomer of claim 42, wherein the amino acid sequence of the mutant protein comprises SEQ ID NO:17.

### Claim 44 (depends on 40)

The mutant platelet factor 4 (PF4) monomer of claim 40, wherein at least 5 amino acids are deleted from the N-terminal end of a wild type PF4 monomer.

### Claim 45 (depends on 40)

The mutant protein of claim 40, wherein the mutant platelet factor 4 (PF4) monomer interferes with wild type PF4 tetramer oligomerization.

### Claim 46 (depends on 40)

A method for treating a subject afflicted with heparin-induced thrombocytopenia (HIT) or coagulation pathologies that lead to thrombus formation, angiogenesis or abnormal cell growth comprising administering to the subject an effective amount of the mutant platelet factor 4 (PF4) monomer of claim 40 so as to interfere with PF4 tetramer oligomerization.

### Claim at position 20

47-48. (canceled)