Antibody polypeptides and uses thereof

Abstract

The present invention provides antibody polypeptides with binding specificity for prostate specific antigen (PSA), wherein the antibody polypeptide comprises (a) a heavy chain variable region comprising the amino acid sequences of SEQ ID NO:1 and SEQ ID NO:2 and SEQ ID NO:3 and/or (b) a light chain variable region comprising the amino acid sequences of SEQ ID NO:4 and SEQ ID NO:5 and SEQ ID NO:6, and wherein the heavy chain variable region and light chain variable region comprise framework amino acid sequences from one or more human antibodies. The invention further provides use of said antibody polypeptides in the diagnosis and treatment of prostate cancer.

Claims

1. An antibody polypeptide with binding specificity for prostate specific antigen (PSA), wherein the antibody polypeptide comprises (a) a heavy chain variable region comprising the amino acid sequences of SEQ ID NO:1 and SEQ ID NO:2 and SEQ ID NO:3; and (b) a light chain variable region comprising the amino acid sequences of SEQ ID NO:4 and SEQ ID NO:5 and SEQ ID NO:6 wherein the heavy chain variable region and light chain variable region comprise framework amino acid sequences from one or more human antibodies, and wherein the PSA is human PSA consisting of the amino acid sequence of SEQ ID NO: 7 or is a mature, active form of human PSA consisting of the amino acid sequence of SEQ ID NO: 24.

2. An antibody polypeptide according to claim 1 comprising or consisting of an intact antibody or an antigen-binding fragment selected from the group consisting of Fv fragments, single chain Fv fragments, disulphide-bonded Fv fragments, Fab fragments, Fab′ fragments and F(ab).sub.2 fragments.

3. An antibody polypeptide according to claim 1 comprising a heavy chain variable region which comprises or consists of the amino acid sequence of SEQ ID NO: 8 and/or a light chain variable region which comprises or consists of the amino acid sequence of SEQ ID NO: 9.

4. An antibody polypeptide according to claim 1 further comprising: a) a heavy chain constant region, or part thereof, optionally wherein the heavy chain constant region is of an immunoglobulin subtype selected from the group consisting of IgG1, IgG2, IgG3 and IgG4; and/or b) a light chain constant region, or part thereof, optionally wherein the light chain constant region is of a kappa or lambda light chain.

5. An antibody polypeptide according to claim 1 comprising a heavy chain constant region which comprises or consists of the amino acid sequence of SEQ ID NO: 10 and/or a light chain constant region which comprises or consists of the amino acid sequence of SEQ ID NO: 11.

6. An antibody polypeptide according to claim 1 comprising a heavy chain which comprises or consists of the amino acid sequence of SEQ ID NO: 12 and/or a light chain which comprises or consists of the amino acid sequence of SEQ ID NO: 13.

7. An antibody polypeptide according to claim 1 wherein the antibody polypeptide is linked, directly or indirectly, to a therapeutic moiety.

8. An antibody polypeptide according to claim 7 wherein the therapeutic moiety is a cytotoxic moiety that comprises or consists of one or more radioisotopes.

9. An antibody polypeptide according to claim 7 wherein the therapeutic moiety is a cytotoxic moiety that comprises or consists of one or more cytotoxic drugs.

10. An antibody polypeptide according to claim 1 wherein the antibody polypeptide further comprises a detectable moiety.

11. An antibody polypeptide according to claim 10 wherein the detectable moiety comprises or consists of a radioisotope.

12. An antibody polypeptide according to claim 7 wherein the therapeutic moiety is joined to the antibody polypeptide indirectly, via a linking moiety.

13. An antibody polypeptide according to claim 12 wherein the linking moiety is a chelator, which is deferoxamine (DFO).

14. A method for the treatment of prostate cancer which expresses prostate specific antigen (PSA) in a human patient, the method comprising the step of administering to a human patient having said cancer a therapeutically effective amount of an antibody polypeptide according to claim 1, wherein said prostate cancer expresses PSA, and wherein the antibody polypeptide comprises an Fc-region and induces antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).

15. A method according to claim 14 wherein the prostate cancer to be treated is non-localised prostate cancer.

16. A method according to claim 15 wherein the prostate cancer to be treated is metastatic prostate cancer, optionally micrometastatic prostate cancer.

17. A method according to claim 16 wherein the metastatic prostate cancer to be treated is metastases of the lymph system; metastases of the bone; and/or metastases within pelvis, rectum, bladder, and/or urethra.

18. A method according to claim 14 wherein the prostate cancer to be treated is castration-resistant prostate cancer (CRPC).

19. An antibody polypeptide according to claim 10 wherein the detectable moiety is joined to the antibody polypeptide indirectly, via a linking moiety.

20. An antibody polypeptide according to claim 19 wherein the linking moiety is a chelator, which is deferoxamine (DFO).

21. A method for the treatment of prostate cancer which expresses prostate specific antigen (PSA) in a human patient, the method comprising the step of administering to a human patient having said cancer a therapeutically effective amount of an antibody polypeptide according to claim 7.

22. A method according to claim 17, wherein the metastases of the bone are of the spine, vertebrae, pelvis, and/or ribs.

Description

(1) The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

(2) FIG. 1: Representative SPECT/CT images of LNCaP xenografts scanned up to 7 days post-injection of .sup.111In-DTPA-h5A10.

(3) FIG. 2: Representative SPECT/CT images of LNCaP xenografts scanned up to 7 days post-injection of .sup.111In-DTPA-m5A10.

(4) FIG. 3: Analysis of the tumour-to-contralateral ratios of m5A10 and h5A10 in the LNCaP xenograft mouse.

(5) FIG. 4: Analysis of the liver-to-tumour ratios of m5A10 and h5A10 in the LNCaP xenograft mouse.

(6) FIG. 5: Biodistribution analysis of .sup.111In-DTPA-m5A10 and .sup.111In-DTPA-h5A10 in the LNCaP xenograft mouse.

(7) The following examples are included to demonstrate particular embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute specific modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

EXAMPLES

Example 1—Cloning of Murine 5A10 Monoclonal Antibody to FAb Format

(8) Reagents

(9) Restriction enzymes were from Fermentas and Promega, alkaline phosphatase from Amersham Pharmacia Biotech and dNTP's and T4 DNA ligase from Fermentas. Primers were from the University of Turku, Department of Biotechnology and from Eurogentec. Qiagen kits were used for DNA purifications.

(10) mRNA Extraction and cDNA Synthesis

(11) mRNA was extracted from 5A10 MAb producing hybridoma cells (2E6 cells; Lilja et al, 1991) with Dynal poly-dT magnetic beads according to procedure described by the manufacturer in “Biomagnetic Techniques in Molecular Biology: Technical handbook” (Dynal A. S, 2nd edition, 1995). cDNA synthesis from the mRNA was performed with Super Script enzyme (Gibco BRL): First, the magnetic particles bound with mRNA were incubated at 42° C. for 2 min in a reaction containing 1× reaction buffer, 0.1 M DTT, 2 mM dNTP's and RNase inhibitor (1.5 μl in a 50 μl total reaction volume). Then, 1 μl of Super Script (200 U) was added and the reaction was proceeded 1 hour in 42° C. After cDNA synthesis, the particles and the reaction mixture were separated by magnetic particle collector, the liquid removed, the particles suspended in TE buffer and incubated at 95° C. for 1 min to release the mRNA. After incubation, particle-bound cDNA was collected, mRNA-containing buffer removed, particles washed with TE and finally suspended in 50 μl of TE for storage.

(12) Amplification of the Antibody Genes from cDNA and Cloning

(13) The N-terminal amino acid sequences of the light and heavy chains were determined by Edman degradation method in the University of Turku, Center of Biotechnology sequencing service. The sequences obtained from the light and heavy chain were DIVMTQS [SEQ ID NO: 18] and EVQLVESG [SEQ ID NO: 19], respectively. Based on the N-terminal amino acid sequences, a database search (SwissProt) was performed to identify corresponding nucleotide sequences among the immunoglobulin germline V-genes of mouse. The complementary regions of the forward PCR primers to clone the heavy and light chains were designed based on the search. Reverse primers were designed to bind CH1 and CL, respectively. Primers also contain the restriction enzyme recognition sites needed for cloning (later underlined).

(14) PCR reactions were done in the following conditions (except changing the primers and templates): 125 μM dNTP's, 0.5 μM forward and reverse primers, 1×Pfu reaction buffer and 2.5 U Pfu DNA polymerase (Stratagene). Amplification was done by 30 cycles of 95° C. 30 s, 55° C. 1 min and 72° C. 1 min 30 s.

(15) Light chain was amplified from cDNA with the following primers:

(16) TABLE-US-00014 5A10-L (5′-CCAGCCATGGCTGACATTGTGATGACCCAGTCTCA-3′, NcoI [SEQ ID NO: 20]) and WO252. (5′-GCGCCGTCTAGAATTAACACTCATTCCTGTTGAA-3′, XbaI [SEQ ID NO: 21])

(17) The light chain and the vector pComb3 containing the genes of an unrelated Fab (Barbas et al., 1991) were digested with NcoI and XbaI (for the vector, partial digestion with NcoI), purified from agarose gel and ligated. The ligation product was transformed into E. coli XL1-Blue to obtain plasmid p5A10-L.

(18) Fd (VH+CH1) chain was amplified with the following primer pair: 5A10-H (5′-CCAGCCATGGCTGAGGTGCAATTGGTGGAGTCTGG-3′, NcoI [SEQ ID NO:22]) and WO267 (5′-CTAGACTAGTACAATCCCTGGGCACAATTTTC-3′, SpeI [SEQ ID NO:23]). Fd chain was cloned into the vector pComb3 to replace the Fd gene of an another Fab carried by the vector. PCR products and vector were digested with NcoI (partial digestion with NcoI for both the vector and the insert) and SpeI (New England Biolabs), purified from agarose gel and ligated. The ligation product was transformed into E. coli XL1-Blue to produce vector p5A10-H.

(19) The 5A10 light chain was digested from the plasmid p5A10-L with SpeI and XbaI and, after gel purification, ligated with the gel purified plasmid p5A10-H also digested with the corresponding enzymes. The ligation product was transformed E. coli XL1-Blue to obtain plasmid pComb3-5A10 containing both the light and Fd chain of the Fab 5A10. To remove phage coat protein gene gIIIp fused to the Fd chain, the pComb3-5A10 was further digested with SpeI and NheI, self-ligated and transformed into E. coli XL1-Blue. The plasmid pComb3-5A10D111 enabled expression of soluble functional Fab 5A10.

REFERENCES

(20) Barbas C F 3rd, Kang A S, Lerner R A, Benkovic S J. (1991) Assembly of combinatorial antibody libraries on phage surfaces: The gene III site. Proc. Nat. Acad. Sci., Vol. 88, pp. 7978-7982 Biomagnetic Techniques in Molecular Biology: Technical handbook. Dynal A. S, 2nd edition, 1995 Lilja H, Christensson A, Dahlén U, Matikainen M T, Nilsson O, Pettersson K, Lövgren T. (1991) Prostate-specific antigen in serum occurs predominantly in complex with alpha 1-antichymotrypsin. Clin Chem. 37(9):1618-25

Example 2—Expression and Purification of a Humanised 5A10 Antibody

(21) HEK293 cells were expanded in to a 2 L suspension culture in FreeStyle 293 Expression Medium (Life Technologies). The cell density was on the day for transfection 1×10.sup.6 cells/ml.

(22) The nucleotide sequences encoding the component heavy or light chains (i.e. SEQ ID NOs: 16 and 17, respectively) were codon-optimized for expression in mammalian cells, synthesized and cloned to IgG expression vectors. The plasmid DNA (expression vector) containing the nucleotide sequences for the heavy and light chains was then mixed with the transfection agent and incubated for 10 min in RT. The DNA-transfection agent-mix was slowly added to cell culture while slowly swirling the flask. The transfected cell culture was then incubated at 37° C., 8% CO.sub.2 on an orbital shaker platform rotating at approx. 135 rpm, for seven days.

(23) Culture medium was harvest by centrifugation and filtered through 5 μm, 0.6 μm and 0.22 μm filter systems.

(24) Antibodies were purified by Protein G chromatography and the buffer was changed to PBS pH 7.4 by dialysis; subsequently, the antibodies were concentrated by ultrafiltration.

(25) Concentration was measured by absorbance.

(26) DNA:

(27) TABLE-US-00015 Light chain: p5A10VLhDhk (4300 bp) amount: 0.35 mg Heavy chain: p5A10VHhDhIgG1 (4900 bp) amount: 0.60 mg

(28) The DNA amounts were not optimized.

(29) Transfection agent: proprietary (however, suitable commercially-available transfection agents are readily available, such as Xfect™ Transfection Reagent (Clontech), Lipofectamine (Life Technologies), FuGENE® HD Transfection Reagent (Promega), FreeStyle™ Max Reagent (Invitrogen), DEAE-dextran, polyethylenimine and calcium phosphate).

(30) Overall yield: 15 mg (7.5 mg/L).

Example 3—Characterisation of h5A10: Affinity

(31) Aims of Study

(32) The aim of the study was to investigate the binding kinetics between eight variants of the antibody 5A10 and the antigen PSA by using the technique of Surface Plasmon Resonance (SPR) on a Biacore™ instrument.

(33) In order to investigate the quality of the protein samples (antibodies and antigen), a SDS-PAGE gel was run prior to the SPR experiments.

(34) In a Pre-Study, different parameters were investigated in order to find the appropriate conditions for the experiments in the Study.

(35) In the Study, multiple binding measurements were performed for the eight antibodies and the antigen. From the collected data, the association and dissociation rate constants (k.sub.on and k.sub.off) and the dissociation constants (KD) were calculated and reported here.

(36) Reagents and Instrument Information

(37) Following solutions of the eight antibodies and one antigen were provided by Diaprost AB: PSA 169 μg/ml m5A10 (141203) 1 mg/ml m5A10 (140905) 4 μM m5A10-DFO (140905) 4 μM m5A10-DOTA (140905) 4 μM m5A10-DTPA (140905) 4 μM h5A10 (141203) 1 mg/ml h5A10-DFO (141203) 2 mg/ml h5A10-DTPA (141203) 1 mg/ml h5A10-DOTA (141211) 2.5 μM

(38) All the samples were aliquoted and kept in −20° C. freezer prior to analysis.

(39) All binding experiments were performed on CM4 chip on a Biacore™ 3000 instrument. The chip and all the reagents needed for activation, immobilization, deactivation, binding and regeneration were purchased from GE Healthcare and used according to the guidelines from the manufacturer.

(40) SDS-PAGE

(41) (a) Description of the Experiment

(42) The reagents provided by Diaprost AB were run on a TRIS-Tricine 10-20% acrylamide gel from Novex according to the guidelines from the manufacturer.

(43) Two series of the protein samples, native and reduced, were run simultaneously on the same gel together with a standard sample.

(44) Each sample in the native series contained: 1-1.3 μg of the protein, TRIS-buffer pH 7.4, SDS and loading buffer.

(45) Each sample in the reduced series contained: 1-1.3 μg of the protein, TRIS-buffer pH 7.4, SDS, loading buffer and 0.04% v/v beta2-merkaptoethanol (the reducing agent).

(46) The staining of the gel was performed in commasie brilliant blue solution of acetic acid, ethanol and water with the corresponding proportions of 0.7, 3.0, 6.3.

(47) The destaining of the gel was performed in the solution of acetic acid, ethanol and water with the corresponding proportions of 0.7, 3.0, 6.3.

(48) (b) Results & Conclusions

(49) It is evident from these results that the antibody and antigen samples are of high quality and purity (results not shown).

(50) Affinity Study

(51) (a) Two Experiments where Performed in Order to Determine the Affinity of the Interactions Using the Below Described Conditions.

(52) Immobilisation of antigen on a CM4 chip.

(53) Activation of the chip CM4-1 was performed according to manufacturer's guidelines for amine coupling using EDC and NHS mixture.

(54) A solution containing 3.00 μg/ml of the antigen PSA (stock solution of PSA diluted in 10 mM NaAc-buffer pH 3.8) was flown over channels fc2-4 on the chip CM4-1 in order to immobilize the antigen to the chip. Flow rate: 5 μl/min, volume: 200 μl.

(55) TABLE-US-00016 Target RU ≤ M.sub.w/10 M.sub.w(PSA) = 30 000 Da Target RU (PSA) ≤3000

(56) Channel fc1 was used as a blank.

(57) The following immobilization was achieved in the first experiment:

(58) TABLE-US-00017 fc2 = 1450 RU fc3 = 850 RU fc4 = 900 RU

(59) The following immobilization was achieved in the second experiment:

(60) TABLE-US-00018 fc2 = 1325 RU fc3 = 890 RU fc4 = 1060 RU

(61) All channels (fc1-4) were blocked by ethanolamine after activation and immobilization.

(62) These data demonstrate that appropriate immobilization was achieved using 3.00 μg/ml of the antigen. (b) Investigation of the Association Phase

(63) The association phase of the eight antibodies to the chip CM4-1 was followed for 5 minutes when solutions of 4 different concentrations of each antibody (stock solutions diluted in HSP-buffer) were flown over the channels fc2-4 on the chip CM4-1 with a rate of 30 μl/min.

(64) The investigated concentrations for each antibody were: 100, 50, 25, and 12.5 nM.

(65) Additionally association data was obtained from the experiments where the dissociation process was followed for 8 hours.

(66) In total, 3-9 individual association experiments for each antibody were performed.

(67) The signal from the blank, fc1, was subtracted for all the data.

(68) We found that after 5 minutes, we were able to fit the data for the association processes. (c) Investigation of the Dissociation Phase

(69) The dissociation phase was followed for 8 hours for each of the antibodies.

(70) The signal from the blank, fc1, is subtracted in all the data used in the calculations of the dissociation rate constant.

(71) The data indicate that the dissociation processes are very slow. (d) Estimation of the Dissociation Rate Constant (k.sub.off)

(72) The dissociation phase data was fitted and the dissociation rate constants (k.sub.off) were estimated (see Table 1). (e) Estimation of the Association Rate Constant (k.sub.on)

(73) In order to estimate the association rate constants, the dissociation rate constants (Table 1) were used in the fitted equations. (f) Estimation of the Dissociation Constant (k.sub.D)

(74) Dissociation constant (K.sub.D) for each of the tested antibodies are shown in Table 1.

(75) TABLE-US-00019 TABLE 1 K.sub.off Antibody (10.sup.−6 s.sup.−1) K.sub.on (10.sup.6 M.sup.−1 s.sup.−1) K.sub.D (10.sup.−11 M) m5A10 (140905) 6.3 0.29 2 m5A10 (141203) 5.4 0.25 2 hu5A10 (141203) 4.8 0.62 1 m5A10-DFO (140905) 6.6 0.29 2 h5A10-DFO (141203) 8.3 0.17 5 m5A10-DOTA (140905) 6.3 0.28 2 h5A10-DOTA (141211) 11.7 0.54 2 m5A10-DTPA (140905) 6.3 0.27 2 h5A10-DTPA (141203) 4.8 0.39 1

(76) The dissociation constants (K.sub.D) are in the 10.sup.−11 M range for all eight antibodies.

(77) Although not statistically significant, the dissociation constant for the humanised antibody appears to be higher than that of the parent murine antibody.

(78) Conjugation of the humanised antibodies does not appear to affect the affinity noticeably since the K.sub.D is not significantly changed.

(79) Summary

(80) The association processes are very fast for all antibodies and the association rate constants (k.sub.on) are all in the 10.sup.5 M.sup.−1 s.sup.−1 range based on 3-9 experiments for each antibody. The dissociation processes are very slow and almost in the range of technical limitations of Biacore™. The dissociation rate constants (k.sub.off) are all in the 10.sup.−6 s.sup.−1 range based on 3-9 experiments for each antibody. The dissociation constants (K.sub.D) are in the 10.sup.−11 M range for all antibodies.

Example 4—Characterisation of h5A10: Aggregation

(81) Executive Summary

(82) Dynamic light scattering (DLS) studies have been carried out on 6 variants of the IgG in order to study their propensity to aggregate. The DLS results show that all constructs have a reasonable size (200 kDa or slightly above 200 kDa assuming a spherical protein) and little or no aggregation.

(83) Objective

(84) To characterise six IgG constructs with respect to oligomeric state using dynamic light scattering.

(85) Results

(86) Dynamic Light Scattering

(87) Dynamic light scattering was measured at 20° C. in duplicate samples using the Malvern Zetasizer APS equipment. Each sample was measured three times. The HBS-EP buffer (from the customer) was used as control to make sure that the buffer was reasonably free from dust and aggregates. No reliable measurements of aggregates in the HBS-EP buffer could be made, which is good, since the buffer should be free from aggregates. All samples could be reliably measured using the number distribution function. The average radius of the most abundant species was calculated along with the polydispersity of the species. The average mass distribution of this species was also calculated, see Table 2.

(88) TABLE-US-00020 TABLE 2 Dynamic light scattering data derived from size distribution Polydispersity Mass distribution Construct Average radius (nm) (%) (%) m5A10-DFO 6.1 22.7 100 h5A10-DFO 6.3 10.2 98.7 m5A10-DOTA 6.2 24.6 100 h5A10-DOTA 6.4 11.3 99.1 m5A10-DTPA 6.2 27.5 100 h5A10-DTPA 6.2 10.6 98.9 Polydispersity = Standard deviation of radius/Average radius × 100%

(89) A radius of 5.7 nm corresponds to a molecular weight of about 200 kDa for a protein having a perfect spherical shape. A radius of 6.1 nm corresponds to a molecular weight of about 230 kDa for a protein having a perfect spherical shape. This is reasonably close to the molecular weight of 150 kDa for IgG molecules, which means that most of the samples primarily consist of monomeric and/or dimeric IgG molecules. The reason for not excluding dimers is that light scattering give a rough size estimate based on molecular shape and this makes it difficult to separate monomers and dimers but easy to separate large aggregates from monomers. Small particles below 1 kDa were found in all mouse IgG samples. These particles have been disregarded in table 1, since it is assumed that they belong to a component found in the buffer due to their small size. On the contrary a small fraction of larger aggregates is found in all human IgG but not in the mouse IgG.

(90) Conclusions

(91) Dynamic light scattering shows that all constructs have a reasonable size and little or no aggregation. The size distributions for all six constructs are overlapping. Surprisingly bufferlike particles (<1 kDa) are found in all mouse IgG samples, whereas larger aggregates are found in the human IgG samples only.

Example 5—Characterisation of h5A10: In Vivo Biodistribution

(92) This study compares in vivo biodistribution of murine 5A10 and human 5A10 when labeled with .sup.111In.

(93) Material and Methods

(94) A humanised counterpart antibody, h5A10, was produced as described below. Antibodies: The exemplary humanised monoclonal antibody 5A10 (IgG1/kappa, transient expressed in HEK 293 cells; see Example 2), comprising a heavy chain according to SEQ ID NO:12 and a light chain according to SEQ ID NO:13, was provided by Innovagen AB, Lund (lot no 90475.33, conc 1.0 mg/ml in PBS, pH 7.4). A non-specific IgG antibody was utilised as an isotype control (IgG antibody from mouse serum, Sigma 1-8765).
Conjugation and Radiolabelling
Conjugation of CHX-A″-DTPA with 5A10:

(95) Solutions of the murine and humanised 5A10 mAbs in PBS or NaCl was adjusted to pH 9.2 using 0.07 M sodium borate buffer (Sigma Aldrich). The protein solution was then conjugated with chelator CHX-A″-DTPA (Macrocyclics, USA) in a molar ratio of 3:1 chelator to antibody at 40° C. for 4 h. The reaction was terminated and CHX-A″-DTPA-11B6 (DTPA-11B6) was separated from free chelate by size-exclusion chromatography on a NAP-5 column (GE Healthcare), equilibrated with 20 ml 0.2 M ammonium acetate buffer, pH 5.5. The conjugated 5A10 antibodies were eluted with 1 ml ammonium acetate buffer and aliquoted samples of 200 uL were stored at −20° C.

(96) Radiolabeling of DTPA-5A10:

(97) Murine and humanised DTPA-5A10, in ammonium acetate buffer pH 5.5 was mixed with a predetermined amount of .sup.111InCl.sub.3 (Mallinkrodt Medical, Dublin, Ireland). After incubation at room temperature for 2 h, the labeling was terminated and purified on a NAP-5 column, equilibrated with PBS (Thermo Scientific, USA). Labelling efficiency and kinetics were monitored by instant thin-layer chromatography (ITLC) (Biodex, Shirley, N.Y., USA) eluted with 0.2 M citric acid (Sigma Aldrich). In this system, the radiolabelled conjugate remains at the origin line, while free .sup.111In. The radioactive distribution was determined using a Cyclone Storage Phosphor System with Optiquant quantification software (Perkin Elmer; Waltham, Mass., USA)

(98) Animal Studies

(99) For in vivo studies, the prostate carcinoma cell lines LNCaP expressing hK2 (ATCC, Manassas, Va., USA) was used. Cells were cultured in RPMI 1640 medium (Thermo Scientific) supplemented with 10% fetal bovine serum and PEST (penicillin 100 IU/ml and 100 μg/ml streptomycin) from Thermo Scientific. The cells were maintained at 37° C. in a humidified incubator with 5% CO.sub.2 and were detached with trypsin-EDTA solution (0.25% trypsin, 0.02% EDTA buffer, Thermo Scientific). Matrigel™ matrix (BD-Biosciences, San-Jose, Calif., USA) was used when xenografting LNCaP cells.

(100) All animal experiments were performed in accordance with national legislation on laboratory animals' protection and with the approval of the Ethics Committee for Animal Research (Lund University, Sweden)

(101) Male immunodeficient nude mice, NMRI-Nu, 6-8 wk old (Janvier Labs, France), were used for this study. All mice were s.c. xenografted with LNCaP cells on their left flank, 5-6 million cells, in 100 μl growth medium and 100 μl Matrigel. The xenografts were allowed to grow for 6-8 weeks.

(102) SPECT/CT Imaging Studies

(103) SPECT/CT imaging studies were performed on .sup.111In-DTPA-h5A10 and .sup.111In-DTPA-m5A10. Animals were anaesthetized with 2% to 3% isoflurane gas (Baxter; Deerfield, Ill., USA) during imaging. NMRI-nu mice with s.c. LNCaP xenografts were intravenously injected in tail vein with .sup.111In-DTPA-h5A10 (n=4) or .sup.111In-DTPA-h5A10 (n=4) with approximately 13-15 MBq, 50 ug of mAb in 100 uL PBS. Animals were imaged for 1 h by using a preclinical SPECT/CT scanner (NanoSPECT/CT Plus, Bioscan; Washington, D.C., USA) with the NSP-106 multi-pinhole mouse collimator. Imaging was performed 1, 2, 3 and 7 days post-injection. SPECT data were reconstructed using HiSPECT software (SciVis; Goettingen, Germany). CT imaging was done before each whole-body SPECT. SPECT/CT images were analysed using InVivoScope 2.0 software (inviCRO; Boston, Mass., USA), and region-of-interest, ROIs, were drawn using the CT image as anatomical reference.

(104) Biodistribution Studies

(105) Biodistribution studies were performed on both .sup.111In-DTPA-h5A10 and .sup.111In-DTPA-m5A10. Groups of animals (n=12) of mice were intravenously injected with .sup.111In-DTPA-h5A10 (approximately 3-4 MBq, 50 ug mAb in 100 uL PBS) or .sup.111In-DTPA-m5A10 (3-4 MBq, 50 ug mAb in 100 uL PBS). The animals were sacrificed 7 days p.i. and organs of interest were collected and analysed with an automated NaI(Tl) well-counter with a 3-inch NaI (Tl) detector (1480 WIZARD, Wallac Oy, Turku, Finland).

(106) The tissue uptake value, expressed as percent injected dose per gram tissue (% IA/g), was calculated as:
% IA/g=(tissue radioactivity/injected radioactivity)/organ weight×100
wherein for iv injections:

(107) $\begin{array}{c}\mathrm{Injected}\mathrm{radioactivity}=\mathrm{Radioactivity}\mathrm{in}\mathrm{the}\mathrm{syringe}\\ —\mathrm{radioactivity}\mathrm{left}\mathrm{in}\mathrm{used}\mathrm{syringe}\\ —\mathrm{radioactivity}\mathrm{in}\mathrm{tail}\end{array}$

(108) The organs were also weighed following dissection. Data were corrected for background and physical decay.

(109) Results

(110) SPECT/CT Imaging

(111) Representative SPECT/CT images of LNCaP xenografts scanned up to 7 days post-injection of .sup.111In-DTPA-h5A10 and .sup.111In-DTPA-m5A10 are shown in FIGS. 1 and 2. The activity rapidly accumulates in the LNCaP tumour within by 24 hours, and the radioactivity remains high at 7 days post-injection. High accumulation of activity was also detected in the liver. The ROI analysis showed that the ratio of tumour to soft tissue of the contralateral leg was 5.2±0.84 for .sup.111In-DTPA-m5A10 and 6.4±1.3 for .sup.111In-DTPA-h5A10 at 7 days p.i.

(112) Biodistribution

(113) To more thoroughly investigate the activity accumulation in different organs, an ex vivo biodistribution was performed in a larger number of animals (n=12 per antibody).

(114) Tumour to contralateral ratios of .sup.111In-DTPA-m5A10 and .sup.111In-DTPA-h5A10 over time are shown in FIG. 3. Unexpectedly, this ratio was observed to be markedly higher for the humanised antibody than for the ‘parent’ murine antibody.

(115) Tumour to liver ratios of .sup.111In-DTPA-m5A10 and .sup.111In-DTPA-h5A10 over time are shown in FIG. 4. Again, unexpectedly, this ratio was observed to be markedly higher for the humanised antibody than for the ‘parent’ murine antibody.

(116) The biodistribution data of .sup.111In-DTPA-m5A10 and .sup.111In-DTPA-h5A10 is shown in FIG. 5.

(117) The biodistribution for the murine and humanized 5A10 in NMRI mice with LNCaP xenografts showed a high tumor accumulation at 7 days while accumulation in other organs, such as bone, muscle were much lower.

(118) Comparison of the biodistribution data for humanised .sup.111In-DTPA-h5A10 with that for the parent murine antibody (.sup.111In-DTPA-m5A10) revealed an unexpected and advantageous difference. The tumor accumulation was significantly much higher for the humanized than for the murine, with 7.4±1.4% IA/g for h5A10 compared to 2.7±0.75% IA/g for m5A10 (p<0.001) at 7 days p.i. (FIG. 6). Accumulation of radioactivity in other organs was approximately on the same level for both antibodies, making tumor-to-organ ratios much higher for the humanized h5A10 than for the parent murine m5A10 (Table 3)

(119) TABLE-US-00021 TABLE 3 Tumor-to-organ ratios h5A10 m5A10 Blood 2.9 1.1 Heart 7.9 3.4 Lung 4.7 2.0 Liver 3.0 0.9 GI tract 21.7 10.8 Kidneys 9.1 3.6 Salivary 5.1 2.7 Skin 4.9 3.0 Muscle 44.2 17.6 Testis 9.1 4.8 Bone 7.3 3.1
Conclusions and Discussion

(120) The results of this study demonstrate the following: the humanised 5A10 antibody, .sup.111In-h5A10, effectively targets prostate tumours in vivo; the humanised h5A10 antibody exhibits an unexpectedly better tumor accumulation than its murine antibody and the humanised h5A10 antibody provides better imaging contrast (as shown with higher tumor-to-organ ratios) than the murine antibody

(121) Taken together, these findings provide compelling evidence of the better targeting properties of humanised 5A10 antibodies in the diagnosis and likely better therapeutic efficacy in the treatment of prostate cancer.

Example 6—Radionuclide Therapy Dosimetry Planning and Treatment of Prostate Cancer in a Patient

(122) For radionuclide therapy (RNT), the radiation source is distributed in the whole and the radioactivity is normally administered systemically as a radiopharmaceutical. The radioactivity distribution depends on the amount of radiopharmaceutical that accumulates over time in different tissues, something which varies between patients (1).

(123) RNT treatment should be based on a prescribed absorbed dose (2). Then first one should perform a pre-therapy study using a tracer amount of the radiopharmaceutical, and determine the tumor and organ absorbed doses. Usually, this information is expressed as a factor describing the organ absorbed dose per unit administered activity, in units of mGy/MBq; D.sup.P.sub.T(organ).

(124) If the therapeutic administration is then given under similar conditions, this factor can be used to determine the activity that needs to be administered in order to deliver a prescribed absorbed dose to a given organ, tissue or tumor (4,6).

(125) In the case of prostate cancer treatment with radiolabelled h5A10 antibodies, a pre-therapy study should be based on .sup.111In imaging with .sup.111In-h5A10. .sup.111In is best suitable for quantitative (planar/SPECT) imaging when then .sup.177Lu is to be the therapeutic radionuclide. When then the D.sup.P.sub.T(organ) is determined the therapy can be given with a therapy activity A.sub.T giving a prescribed therapy effect. During therapy, the activity distribution and corresponding dose rate should be calculated based on imaging to get the actual therapy absorbed dose given to tumor and normal organs, necessary for evaluation of treatment.

(126) In case of therapy where the bone marrow toxicity level is reached as a result of the treatment planning then bone-marrow support is necessary and based on dosimetry calculations for the bone marrow cavity the time for reinfusion of stem cells has to be determined.

(127) In summary, the following treatment scheme should be planned accordingly:

(128) Pre-Therapy Dosimetry Study

(129) 1. .sup.111In-labeled h5A10 (200-300 MBq) injection 2. Blood sampling—activity concentration in blood and plasma determined first week. 3. Imaging (SPECT/Planar) over at least 1 week (3-5 times) 4. Organ Dosimetry based on LundaDose scheme (3) or equivalent program 5. Therapy activity determined limited by specified absorbed dose to radiosensitive organs as bone marrow (2-3 Gy), kidneys (20-30 Gy) and liver (12-36 Gy).
Therapy Including Intra-Therapy Dosimetry 1. .sup.177Lu-labeled h5A10 administered (based on pretherapy dosimetry) 2. Blood sampling—activity concentration in blood and plasma 3. Imaging over at least 1 week (3-5 times) 4. Organ Dosimetry based on LundaDose scheme (3) or equivalent program=>Verification of prescribed therapy absorbed dose.
Specific Comments on Dosimetry

(130) The cumulated activity is the number of decays that occur in a given region over a period of time. The unit is Bq s, or Bq h. When ionizing radiation travels through matter, it interacts and deposits energy. The energy imparted is the sum of all energy deposits in a given volume. The absorbed dose is the ratio of the energy imparted and the mass of the volume. The unit of absorbed dose is Gray (Gy), 1 Gy equals 1 J/kg.

(131) From the values of the activity in a tissue at different times, the cumulated activity is determined by integration, and the absorbed dose can be determined. Activity measurements are made using planar imaging for whole-organ dosimetry. Quantitative SPECT/CT allows for dosimetry in smaller volumes using voxel-based methods.

(132) From the 3D distribution of activity concentration values, the absorbed dose rate distribution can be calculated using so-called point dose kernels or voxel S values, describing the energy deposition pattern around a point source located in tissue. This method assumes that the anatomical region is homogeneous in terms of density, such as soft tissues within the trunk. For body regions where the density is heterogeneous, as in the lungs, a direct Monte Carlo calculation is preferable. Here, the activity distribution from SPECT or PET is used as input to a Monte Carlo dose calculation code.

REFERENCES

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