Tumor localization and therapy with labeled antibody fragments specific to tumor-associated markers

• US 4,331,647 A
• Filed: 03/03/1980
• Issued: 05/25/1982
• Est. Priority Date: 03/03/1980
• Status: Expired due to Term

• Current Assignee: Milton D., Lexington Goldenberg
• Original Assignee: Milton D., Lexington Goldenberg
• Inventors: Goldenberg, Milton D.
• Primary Examiner(s): Nucker, Christine M.
• Application Number:
  US06/126,263
• Time in Patent Office: 813 Days
• Field of Search: 424/119
• US Class Current: 424/1.37
• CPC Class Codes: A61K 2121/00 Preparations for use in therapy
• CPC Class Codes: A61K 2123/00 Preparations for testing in vivo
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 51/1048 the tumor cell determinant being a carcino embryonic antigen
• CPC Class Codes: C07K 16/3007 Carcino-embryonic Antigens

Abstract

Methods are provided for using radiolabeled antibody fragments specific to tumor-associated markers for detection, localization and therapy of tumors. Mixtures of labeled fragments with varied specificity or multivalent hybrid fragments permit detection and localization of more than one tumor or tumor cell type. Antibodies and injectable compositions for use in the methods of the invention are also provided.

Claims

1. A method for detecting and localizing a tumor which either produces or is associated with a cytoplasmic, intracellular or cell-surface marker substance which comprises injecting a human subject parenterally with a marker-specific fragment obtained by cleavage of an antibody specific to said marker substance and radiolabeled with a pharmacologically inert radioisotope capable of detection using a photoscanning device, and subsequently scanning with said device to detect and locate the site or sites of uptake of said labeled antibody fragment by said tumor.

2. The method of claim 1, wherein said market substance is an oncofetal antigen.

3. The method of claim 1, wherein said market substance is a placental antigen.

4. The method of claim 1, wherein said marker substance is an oncogenic or tumor or tumor virus-associated antigen.

5. The method of claim 1, wherein said marker substance is a tissue- or organ-associated antigen, an ectopic hormone or a normal antigen or variant thereof.

6. The method of claim 1, wherein said marker substance is carcinoembryonic antigen (CEA).

7. The method of claim 1, wherein said marker substance is alphafetoprotein (AFP).

8. The method of claim 1, wherein said marker substance is human chorionic gonadotropin (HCG) or the beta-subunit thereof.

9. The method of claim 1, wherein said marker substance is colon-specific antigen-p (CSAp).

10. The method of claim 1, wherein said market substance is one of prostatic acid phosphatase, pancreatic oncofetal antigen, placental alkaline phosphatase, pregnancy beta₁ -globulin, parathormone, calcitonin, tissue polypeptide antigen, T-antigen, beta₂ -microglobulin, mammary tumor-associated glycoproteins (MTGP), galactyosyl transferase-II (GT-II), gp-52 viral-associated antigen, ovarian cystadenocarcinoma-associated antigen (CA-58, CCA, TA-4), basic fetoprotein (BFP), terminal deoxynucleotidyl transferase (TdT), cytoplasmic melanoma-associated antigens, human astrocytoma-associated antigen (HAAA), common glioma antigen (CGA), glioembryonic antigen (GEA), glial fibrillary acidic protein (GFA), common meningioma antigen (CMA), ferritin, and tumor angiogenesis factor (TAF).

11. The method of claim 1, wherein said marker-specific fragment is an Fab, Fab'"'"', and F(ab'"'"')₂ fragment obtained by cleavage of said specific antibody.

12. The method of claim 1, wherein said radioisotope is one of Iodine-131, Iodine-123, Iodine-126, Iodine-133, Bromine-77, Indium-111, Indium-113m, Gallium-67, Gallium-68, Ruthenium 95, Ruthenium-97, Ruthenium-103, Ruthenium-105, Mercury-107, Mercury-203, Rhenium-99m, Rhenium-105, Rhenium-101, Tellurium-121m, Tellurium-122m, Tellurium-125m, Thulium-165, Thulium-167, Thulium-168, Technetium-99m and Fluorine-18.

13. The method of claim 1, wherein said radioisotope is Iodine-131 or Iodine-123.

14. The method of claim 1, wherein the amount of radiolabel introduced by direct chemical substitution into said marker-specific antibody fragment is sufficient to reduce its specific market substance immunoreactivity by from 5 to 33%.

15. The method of claim 1, wherein said marker-specific antibody fragment is substantially monospecific for the marker substance, having a crossreactivity with other antigens of less than 15% and an immunoreactivity for the marker substance of more than 50%.

16. The method of claim 1, wherein the specific antibody cleaved to produce said marker-specific fragment is a monoclonal antibody having a marker substance specificity of at least 90%.

17. The method of claim 1, wherein said fragment is labeled with a radioisotope of iodine, an average of at least 2.5 atoms of iodine per fragment molecule being introduced and no more than will reduce the antibody specificity by more than 33%.

18. The method of claim 1, wherein said tumor is located in the brain and said fragment is injected intravenously, intraarterially or intrathecally.

19. The method of claim 18, wherein the fragment is injected intravenously.

20. The method of claim 1, wherein prior to photoscanning, a second substance is injected into the subject, said second substance being radiolabeled with a radioisotope emitting at a different energy from the antibody fragment label and capable of independent detection by said photoscanning device, the level of activity of said second substance being used to determine the background activity due to non-targeted specific antibody fragment, said background activity being subtracted from the total activity of the specific antibody fragment, whereby the activity of substantially only the targeted, tumor-associated antibody fragment is determined.

21. The method of claim 20, wherein said second substance is technetium-99m-labeled normal human immunoglobulin, technetium-99m-labeled human serum, technetium-99m-sulfur colloid, technetium-99m-pertechnetate, or a combination thereof.

22. The method of claim 20, wherein said second substance is the corresponding fragment obtained by analogous cleavage of normal immunoglobulin from the same or different species as that used to prepare said specific antibody.

23. The method of claim 22, wherein said corresponding fragment is labeled with a different isotope of the same element used to label the fragment of said specific antibody.

24. The method of claim 23, wherein the specific antibody fragment is labeled with one of a pair of radioisotopes, and the corresponding normal immunoglobulin fragment is labeled with the other of the pair, said pair being Iodine-131 and Iodine-123;

Indium-111 and Indium-113m;

Gallium-67 and Gallium-68;

Ruthenium-97 and Ruthenium-103;

or Mercury-197 and Mercury-203.

25. A method for detecting and localizing a tumor which either produces or is associated with a cytoplasmic, intracellular or cell-surface marker substance, which comprises injecting a human subject parenterally with two marker-specific fragments, one fragment being a marker-specific fragment obtained by cleavage of a first antibody specific to a first tumor associated marker and the second fragment being a marker-specific fragment obtained by cleavage of a second antibody specific to a second, different tumor-associated marker, each of said fragments being radiolabeled with a pharmacologically inert radioisotope capable of detection using a photoscanning device, and subsequently scanning with said device to detect and locate the site or sites of uptake of one or both of said labeled fragments by said tumor.

26. The method of claim 25, wherein said two marker-specific fragments are obtained by cleavage of two of anti-CEA antibody, anti-AFP antibody, anti-HCG antibody and anti-CSAp antibody.

27. The method of claim 26, wherein said two fragments are labeled with different radioisotopes.

28. The method of claim 25, wherein prior to photoscanning, a reference substance is injected into the subject, said reference substance being radiolabeled with a radioisotope emitting at a different energy from either antibody fragment label and capable of independent detection by said photoscanning device, the level of activity of said reference substance being used to determine the background activity due to non-targeted specific antibody fragment, said background activity being subtracted from the total activity of each specific antibody fragment, whereby the activity of substantially only targeted, tumor-associated antibody fragments is determined.

29. The method of claim 28, wherein said reference substance is technetium-99m-labeled normal human immunoglobulin, technetium-99m-labeled human serum, technetium-99m-sulfur colloid, technetium-99m-pertechnetate, or a combination thereof.

30. The method of claim 28, wherein each of said two fragments is labeled with a different radioisotope;

a fragment obtained by analogous cleavage of normal immunoglobulin from the same or different species used to prepare each said specific antibody and corresponding to each of said two fragments is used as a reference substance;

each said reference fragment is labeled with a different isotope of the same element used to label the corresponding fragment of said specific antibody; and

the activity of the reference fragment corresponding to each of said two fragments is used to determine the background activity and the targeted fragment activity for the specific antibody fragment to which it corresponds.

31. A method for detecting and localizing a tumor which either produces or is associated with a cytoplasmic, intracellular or cell-surface marker substance, which comprises injecting a human subject parenterally with an F(ab'"'"')₂ hybrid formed from two chemically joined Fab'"'"' fragments, said fragments being a first marker-specific Fab'"'"' fragment obtained by cleavage of an antibody specific to a first tumor-associated marker and a second, different marker-specific Fab'"'"' fragment obtained by cleavage of a second antibody specific to the same or different tumor-associated marker, said hybrid being radiolabeled with a pharmacologically inert radioisotope capable of detection using a photoscanning device, and subsequently scanning with said device to detect and locate the site or sites of uptake of said labeled hybrid by said tumor.

32. The method of claim 31, wherein said hybrid is obtained by cleavage and recombination of a mixture of F(ab'"'"')₂ fragments specific to each said specific antibody.

33. The method of claim 31, wherein said hybrid contains a marker-specific Fab'"'"' arm obtained by cleavage of anti-CEA antibody, anti-AFP antibody, anti-HCG antibody, or and anti-CSAp antibody.

34. The method of claim 31, wherein said hybrid contains marker-specific Fab'"'"' arms obtained by cleavage of two different antibodies selected from anti-CEA antibody, anti-AFP antibody, anti-HCG antibody and anti-CSAp antibody.

35. The method of claim 31, wherein prior to photoscanning, a second substance is injected into the subject, said second substance being radiolabeled with a radioisotope emitting at a different energy from the hybrid label and capable of independent detection by said photoscanning device, the level of activity of said second substance being used to determine the background activity due to non-targeted hybrid, said background activity being subtracted from the total activity of the hybrid, whereby the activity of substantially only the targeted, tumor-associated hybrid is determined.

36. The method of claim 35, wherein said second substance is technetium-99m-labeled normal human immunoglobulin, technetium-99m-labeled human serum, technetium-99m-sulfur colloid, technetium-99m-pertechnetate, or a combination thereof.

37. The method of claim 35, wherein said second substance is an F(ab'"'"')₂ fragment obtained by analogous cleavage of normal immunoglobulin from the same or different species as that used to prepare either said antibody specific to a first tumor-associated marker or said second antibody specific to the same or different tumor-associated marker.

38. The method of claim 37, wherein said second substance is labeled with a different isotope of the same element used to label the F(ab'"'"')₂ hybrid.

39. The method of claim 38, wherein the F(ab'"'"')₂ hybrid is labeled with Iodine-131 or Iodine-123 and the second substance is labeled with the other of Iodine-131 or Iodine-123.

40. A method of tumor radiotherapy, which comprises parenterally injecting into a human subject having a tumor which produces or is associated with a cytoplasmic, intracellular or cell-surface marker substance an effective tumor reducing amount of a marker-specific fragment obtained by cleavage of an antibody specific to said marker substance and radiolabeled with a pharmaceutically inert, radiotherapeutically effective radioisotope.

41. A method of tumor radiotherapy, which comprises parenterally injecting into a human subject having a tumor which either produces or is associated with at least one cytoplasmic, intracellular or cell-surface marker substance an effective tumor reducing amount of an F(ab'"'"')₂ hybrid formed from two chemically joined Fab'"'"' fragments, said fragments being a first marker-specific Fab'"'"' fragment obtained by cleavage of an antibody specific to a first tumor-associated marker and a second, different marker-specific Fab'"'"' fragment obtained by cleavage of a second antibody specific to the same or different tumor-associated marker, said hybrid being radiolabeled with a pharmacologically inert, radiotherapeutically effective radioisotope.

42. The method of claim 41, wherein said F(ab'"'"')₂ hybrid is prepared by the steps of pepsin-catalyzed cleavage of anti-CEA antibody to produce an F(ab'"'"')₂ fragment and reductive cleavage thereof to form CEA-specific Fab'"'"' fragments, pepsin-catalyzed cleavage of anti-CSAp antibody to produce an F(ab'"'"')₂ fragment and reductive cleavage thereof to form CSAp-specific Fab'"'"' fragments, and oxidative coupling of a mixture of said CEA-specific Fab'"'"' fragments and sad CSAp-specific Fab'"'"' fragments to form F(ab'"'"')₂ fragments, at least a portion of which are F(ab'"'"')₂ hybrids specific to both CEA and CSAp.

43. The method of claim 42, wherein said F(ab'"'"')₂ hybrid is purified by sequential affinity chromatography through a column containing bound CEA and a column containing bound CSAp.

44. An injectable composition, which comprises:

(a) a marker-specific fragment obtained by cleavage of an antibody specific to a cytoplasmic, intracellular or cell-surface marker substance which is provided or associated with a tumor, said fragment being radiolabeled with a pharmacologically inert radioisotope capable of detection using a photoscanning device; and

(b) a pharmaceutically acceptable injection vehicle.

45. The composition of claim 44, which further comprises a second marker-specific fragment obtained by cleavage of a second antibody specific to a second tumor-associated marker, said fragment being radiolabeled with a pharmacologically inert radioisotope capable of detection using a photoscanning device.

46. The injectable composition of claim 44, which further comprises a reference fragment which is the corresponding fragment obtained by analogous cleavage of normal immunoglobulin from the same or different species as that used to prepare said specific antibody, said reference fragment being radiolabeled with a different isotope of the same element used to label said marker-specific fragment and emitting at an energy capable of independent detection using said photoscanning device.

50. The injectable composition of claim 44, wherein said marker-specific fragment further contains in chemical combination an addend containing at least five atoms of boron with at least the natural abundance of Boron-10 isotope.

47. An injectable composition which comprises:

(a) an F(ab'"'"')₂ hybrid formed from two chemically joined Fab'"'"' fragments, said fragments being a first marker-specific Fab'"'"' fragment obtained by cleavage of an antibody specific to a first tumor-associated marker and a second different marker-specific Fab'"'"' fragment obtained by cleavage of a second antibody specific to the same or different tumor associated marker, said hybrid being radiolabeled with a pharmacologically inert radioisotope capable of detection using a photoscanning device; and

(b) a pharmaceutically acceptable injection vehicle.

48. The injectable composition of claim 47, which further comprises a reference F(ab'"'"')₂ fragment from normal immunoglobulin from the same or different species as that used to prepare said hybrid, said reference fragment being radiolabeled with a different isotope of the same element used to label said F(ab'"'"')₂ hybrid and emitting at an energy capable of independent detection using said photoscanning device.

49. A method of tumor radiotherapy, which comprises parenterally injecting into a human subject having a tumor which produces or is associated with a cytoplasmic, intracellular or cell-surface marker substance an effective tumor reducing amount of a marker-specific fragment obtained by cleavage of an antibody specific to said marker substance and radiolabeled with a pharmacologically inert radioisotope capable of detection with a photoscanning device, said labeled fragment further containing in chemical combination an addend containing at least five atoms of boron with at least a natural abundance of Boron-10 isotope;

wherein the location of said tumor is determined using said photoscanning device, and a beam of thermal neutrons is then directed at said tumor location.