WO2003097075A1

WO2003097075A1 - Blood treatment

Info

Publication number: WO2003097075A1
Application number: PCT/GB2003/002160
Authority: WO
Inventors: Ajay K. Kakkar; Guy Nash
Original assignee: Thrombosis Research Institute
Priority date: 2002-05-20
Filing date: 2003-05-20
Publication date: 2003-11-27
Also published as: GB0211515D0; AU2003242826A1

Abstract

Application relates to methods of modifying stored or manipulated blood and blood products in order to counter the increased angiogenic activity resulting from such storage and manipulation. The angiogenic balance may be modified by depletion of accumulated angiogenic factors such as VEGF and bFGF, and/or addition of anti-angiogenic factors such as endostatin. Also disclosed are modified blood products.

Description

BLOOD TREATMENT

Background to the invention

Tumours rely upon an adequate blood supply in order to grow. The development of new vasculature depends upon angiogenic stimulation, which is controlled by the local balance of soluble angiogenic and anti-angiogenic factors. The excision of a primary malignancy has been shown to alter the balance of angiogenic factors released in association with the tumour suggesting that accelerated metastatic growth may be explained by angiogenic factors outweighing anti-angiogenic factors, which, in turn, may be related to the half-lives of tumour-associated factors (O'Reilly et al 1994, Cell 79:315). Angiogenesis is also an important factor in both the likelihood of a primary tumour shedding micrometastases into the circulation and whether such micrometastases successfully penetrate the endothelium and establish themselves at distant sites. In this context the angiogenic factors vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) have been shown to be important (Dirix et al 1997, Brit J Cancer 76:238). A significant factor on the anti-angiogenic side of the balance is endostatin (O'Reilly et al ,op cit.).

For over twenty years, there has been a demonstrated association between perioperative blood transfusion and increased cancer patient mortality (Gantt 1981 , Lancet 2:363). Cancer patients who have had a seemingly curative resection and have received any form of blood transfusion have a lower long-term survival, though the cause of this increased mortality is still unclear. Micrometastases, demonstrated as circulating perioperatively, have been observed to grow in transfused patients leading to cancer recurrence (Maniwa et al 1998, Chest 114: 1668).

An immunomodulatory effect has been hypothesised to explain the observed increased cancer recurrence. Both leukocyte-depleted and buffy coat-depleted blood have been shown to be associated with reduced survival, although this does not appear to be associated with a recurrence of cancer (van der Watering 2001 , Brit J Surg 88:267, Houbiers et al 1994, Lancet 344:573). Transfusion with buffy-coat- depleted blood (still containing a proportion of leukocytes) appears to be associated with more post-operative complications not related to recurrence of cancer than transfusion with leukodepleted blood (containing very few leukocytes) (Jensen et al 1996, Lancet 348: 841). However, in animal models with established tumours, metastasis has been demonstrated to be significantly reduced by administering leukodepleted blood compared to buffy coat -depleted blood (Reviewed in Blajchman 1998, Vox Sang 74 (suppl 2): 315S). On the basis of the published data, therefore, it is unclear whether the effect of blood transfusion on cancer proliferation can be explained by the current immunological model or by some other unknown mechanism or by a combination of more than one such factor, or whether the advantage associated with leucodepletion can be attributed solely to immunomodulation or to some other effect.

Although blood transfusion is frequently a life-saving procedure for many surgical procedures, and the treatment of cancer in general would be difficult or impossible without it, it is nevertheless associated with a significant long-term risk when administered to cancer patients, and possibly others. There is, therefore, a need to establish a mechanism to explain the observed adverse effect and to develop a means of transfusing cancer patients without an increased risk of recurrence or metastasis.

Summary of the invention

Disclosed herein are data demonstrating that blood that has been stored provides an increased angiogenic stimulus as compared with fresh blood. Further, stored blood has increased available levels of angiogenic factors, as exemplified by VEGF and bFGF, and lower available levels of anti-angiogenic factors, as exemplified by endostatin. This shift in the angiogenic balance results in an increased angiogenic stimulus resulting from exposure to stored blood leading to an increased risk of accelerating metastatic growth perioperatively. Further, it is disclosed that angiogenic factors, as exemplified by VEGF and bFGF may be efficiently depleted from or counteracted in stored blood and that such depletion reduces the observed increased angiogenic effect of stored blood to that of fresh blood.

Depletion of active angiogenic factors may be achieved in a number of ways, including specific binding by cognate ligands, such as antibodies, and removal of the complexes by binding to an insoluble matrix. Alternatively, such factors may be neutralised by binding of antibodies, or other cognate ligands. For instance, soluble VEGF receptor (sVEGFR-1 , sFlt-1) is a naturally-occurring competitive inhibitor of VEGF. It is also suggested that the concomitant use of anti-thrombotic / anticoagulant agents that modify the activity of coagulant serine proteases may effect angiogenic activity directly or by up- or down-regulating expression of receptors for other pro-or anti-angiogenic factors or coagulation proteases.

Alternatively, cells responsible for the production and accumulation of angiogenic factors may be removed from blood prior to storage. For example, bFGF release from platelets, or VEGF from leukocytes, into stored blood mat be prevented by removal of the appropriate cells.

As used herein, "stored blood" means blood not used immediately after donation. It includes blood that has been prepared for storage according to standard methods well-known in the art and/or stored for at least 24 hours. Preferably the stored blood has been stored for at least 5 days, more preferably 15 days.

However, the invention is not restricted to blood that has been stored between donation and use. It also relates to autologous blood transfusions where the blood cells may be activated, resulting in accumulation of pro-angiogenic factors and/or depletion or consumption of anti-angiogenic factors during a process of blood preparation. It is equally applicable to blood that has passed through external circuits (such as a cell saver or heart/lung bypass machine) resulting in such accumulation of pro-angiogenic and /or depletion of anti-angiogenic factors. Blood or cells that have been subjected to such ex vivo manipulation are defined as "manipulated".

"Blood product" means any product obtainable from blood, including whole blood, with or without addition of anticoagulants such as citrate, heparin or lepirudin; plasma; serum; preparations of cells such as buffy coat cells, leukodepleted packed cells, erythrocytes, leukocytes or platelets; more highly purified cell preparations such as neutrophils, lymphocytes (T and B combined, or separated), monocytes or macrophages; or highly processed products such as clotting factors.

"Angiogenic factor" includes any factor that tends to promote endothelial growth and /or growth of blood vessels. Examples include molecules such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF, but especially basic fibroblast growth factor or bFGF), interleukin-8 (IL-8), angiogenin, angiotropin, epidermal growth factor (EGF), platelet-derived growth factor (PDGF), transfroming growth factor α (TGF-α), and transforming growth factor β (TGF-β).

"Anti-angiogenic factor" includes any factor that tends to inhibit endothelial growth and/or growth of blood vessels, or otherwise antagonise an angiogenic factor.

Examples include molecules such as endostatin, angiostatin and thrombospondin, and antithrombotic /anticoagulant agents such as heparin, low molecular weight heparin, dermatan sulphate, heparan sulphate, any activated coagulation serine protease inhibitor, including, for example, direct thrombin inhibitors, pentasaccharide, direct factor Xa inhibitors, factor Vila/tissue factor inhibitor and direct and indirect platelet inhibitors.

"Endogenous" referring to a factor means one that is naturally present in the human body. It includes the corresponding recombinant factor having the same primary structure, and fragments thereof having equivalent functional properties. "Affinity purification" means a form of adsorption chromatography in which the molecule to be purified is specifically bound by a complementary binding substance (ligand), which, either before or after this step, is immobilised on an insoluble support (matrix). This is a standard laboratory technique well-known to one of appropriate skill in the art (See "Affinity Chromatography: principles and methods, Pharmacia LKB Biotechnology, Uppsala, Sweden).

"Immunoaffinity purification" means a form of affinity chromatography in which the ligand, as described above, is an antibody or functional fragment of an antibody. It is also a standard laboratory technique, well-known in the art.

"Cancer" as used herein means any neoplastic or proliferative condition and includes any carcinoma, sarcoma, lymphoma, leukaemia, myelodysplasia, tumour of embryonic origin, hepatoma, tumour of the central nervous system or surrounding structures, tumour of the breast, or gastrointestinal, respiratory, renal, cardiovascular, musculoskeletal, endocrine, genitourinary, reticuloendothelial systems or of connective tissue.

Accordingly, disclosed herein is a method of modifying a blood product comprising altering the balance of angiogenic and anti-angiogenic factors therein, such that after storage or manipulation, said blood product has reduced angiogenic activity as compare to unmodified blood.

In one aspect, the invention relates to a method of modifying a blood product , characterised in that said blood product has been stored or manipulated and that the level of an active angiogenic factor therein is depleted. Preferably said angiogenic factor is VEGF. Alternatively, said angiogenic factor is bFGF.

Alternatively, it relates to a method of modifying a stored or manipulated blood product by means of the depletion of two or more angiogenic factors therein. Preferably said factors include VEGF and bFGF. Preferably said depletion of one or more angiogenic factors is by means of affinity purification, more preferably by immunoaffinity purification, and most preferably said immunoaffinity purification is by means of one or more monoclonal antibodies or functional fragments thereof.

In another aspect, the invention relates to a method of modifying a stored or manipulated blood product whereby the level of at least one active angiogenic factor is depleted by means of the addition of one or more antagonists or anti-angiogenic factors. Alternatively, two or more active angiogenic factors are depleted.

Preferably, said antagonist or angiogenic factor binds to and inactivates said angiogenic factor. More preferably said antagonist binds specifically to said factor. Further preferably, said antagonist comprises a soluble receptor or functional fragment thereof. Most preferably, it comprises a soluble FGF or VEGF receptor or functional fragment thereof. Alternatively, it is an antibody or functional fragment thereof.

In another aspect, the invention relates to a method modification of stored or manipulated blood as described above characterised by the addition of one or more anti-angiogenic factors. Preferably, the one or more anti-angiogenic factors added are endogenous to said blood product. Alternatively, they may be exogenous factors not normally present in blood. Preferably said one or more anti-angiogenic factors are recombinant. Alternatively said factor or factors may be of non-human origin. Alternatively said factor or factors may be modified human factors. Such modification may include truncation (possibly by proteolytic cleavage), glycosylation or deglycosylation (either complete or partial), and addition of other modifying groups by covalent or other attachment. Most preferably, one of said one or more anti-angiogenic factors is endostatin. Another aspect of the invention is a blood product prepared according any one of the above methods. Preferably, such product is characterised in that the level of an active angiogenic factor is depleted and /or the level of an anti-angiogenic factor is raised (ie the ratio between angiogenic and anti-angiogenic factors is shifted in favour of anti-angiogenic factors). Preferably, the active angiogenic factor depleted is VEGF. Alternatively, it is bFGF. Alternatively, two or more angiogenic factors are depleted.

In one embodiment, said blood product contains an angiogenic factor bound to an antagonist. Preferably, said antagonist is a soluble receptor or functional fragment thereof. More preferably, it is a soluble VEGF or FGF receptor or functional fragment thereof.

Ina further aspect, said blood product is characterised in that it comprises an anti- angiogenic factor distinguishable from nature-identical factors. Alternatively, it may be characterised by the addition of one or more anti-angiogenic factors. Preferably, the one or more anti-angiogenic factors added are endogenous to said blood product. Alternatively, they may be exogenous factors not normally present in blood. Preferably said one or more anti-angiogenic factors are recombinant. Alternatively said factor or factors may be of non-human origin. Alternatively said factor or factors may be modified human factors. Such modification may include truncation (possibly by proteolytic cleavage), glycosylation or deglycosylation (either complete or partial), and addition of other modifying groups by covalent or other attachment. Most preferably, one of said one or more anti-angiogenic factors is endostatin.

In one embodiment, said blood product is characterised in that it is whole blood. Alternatively, it is leukodepleted blood. In another embodiment, it is an essentially cell-free blood product.

Also provided are any of the blood products described above, for use as a medicament, preferably for the treatment of cancer. Also provided is of any of the blood products described above for the manufacture of a medicament, preferably for the treatment of cancer.

In another aspect the invention provides a method of treatment comprising administering to a patient a blood product prepared according to any of the methods described above. Preferably, the method comprises a pe operative transfusion of blood or any other blood product administered to a patient suffering from cancer. Preferably, the method of treatment is applied to a patient suffering from cancer.

In a further aspect the invention provides a pharmaceutical composition comprising a blood product prepared according to any of the methods described above, which may be administered at the time of transfusion or independently of it..

Also provided is a kit for the depletion of angiogenic factors from a blood product, comprising ligands capable of specific binding to one or more angiogenic factors and a means of binding and removing from solution complexes formed between said angiogenic factors and said ligands. Preferably, said ligands are antibodies or functional fragments thereof. More preferably they are monoclonal antibodies or functional fragments thereof.

In another aspect is provided a sterile vessel containing the blood product as previously described. Preferably, the vessel is in the form of a bag. Alternatively, it may be a bottle, flask, tube or other container adapted for the purpose, made from plastic, glass or other material. Preferably said vessel contains an anti-angiogenic factor. Alternatively, it contains an angiogenic factor antagonist. More preferably, said anti-angiogenic factor or antagonist is immobilised to the inner surface of said vessel. Detailed description of the invention

The invention is described through Examples with reference to the accompanying Tables and Figures, wherein:

Figure 1 shows the levels of circulating bFGF in the plasma of control (benign) and cancer patients compared with 3 week old stored blood.

Figure 2 shows the effect of 1 week storage of platelet-rich plasma on bFGF levels. Figure 3 shows the proliferative effect of 3 week old stored blood on endothelial cells. Figure 4 shows the proliferative effect of plasma from control patients and cancer patients, as compared with 3 week old stored blood, on endothelial cells. Figure 5 shows the proliferative effect of plasma from control patients and cancer patients, as compared with 3 week old stored blood, on CACO-2 cells. Figure 6 shows the formation of microvessels in an angiogenesis assay (CD31 immunostaining, light microscopy x10).

Figure 7 shows the effect of 3 week old stored blood and stored blood depleted of VEGF on angiogenesis.

Examples

Example 1

METHODS

Measurement of angiogenic factors in blood

The circulating endogenous antiangiogenic factor, endostatin as well as angiogenic factors bFGF and VEGF were measured in 21 patients with solid tumours (predominantly gastrointestinal) in comparison to 21 control subjects. Blood was drawn from preoperative cancer patients and age-matched healthy controls, who denied aspirin or non-steroidal anti-inflammatory ingestion. Each citrated sample was promptly centrifuged at 3000 rpm for 10 minutes to collect platelet rich plasma. bFGF, VEGF and endostatin were measured using standard ELISAs in each blood sample.

To test this hypothesis, we collected blood products as supplied by the blood bank, and measured VEGF in each sample. Fresh citrated blood, packed blood cells, buffy coat residue, platelet concentrates and pooled plasma were kept at 4°C. Every 72 hours 20 ml of each blood product was centrifuged at 3000 rpm for 10 minutes. The supernatant was aspirated and stored at -70°C. At the end of one week storage for plasma and platelets, and three weeks storage for blood, a standard ELISA was utilised to measure endostatin, basic fibroblast growth factor (bFGF) and VEGF in the serial plasma samples. After ethical consent was granted, this was compared to age-matched endostatin, bFGF and VEGF measured from 21 healthy controls and 21 patients with solid cancers.

Cell proliferation studies

Since several studies have demonstrated colorectal cancer as being affected by this transfusion related increased mortality, we investigated the mitotic stimulation of a colorectal cancer cell line (CACO-2) with plasma from the above cancer and control patients. CACO-2 forms moderately-differentiated adenocarcinoma when cultured in EMEmedium, 2mM glutamine, 1 % non-essential amino acids (NEAA) and 10 % fetal bovine serum. Mature first passage HUVECs were incubated for 48 hours in Medium 199, 10% fetal calf serum with either 20% plasma from benign or malignant patients, or 20% stored blood from the national blood bank (20 different units of blood were used on the day of their expiry).

The proliferation of both endothelial cells and CACO-2 cells was measured 48 hours after addition of plasma or supernatant from stored blood to wells at approximately 60% confluence. 3-[4,5dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[-4- sulfophenyl]-2H-tetrazolium salt (MTS) assay was used in cell quantification adding 20μL per 200μL medium for approximately 2 hours. MTS is bioreduced in the mitochondria of cells to a coloured formazan product that is measured at 490 nm upon a Molecular Devices plate reader. Optical densitometry coupled to a personal computer allows examination of up to 96 wells simultaneously. The quantity of coloured product is directly proportional to the number of living cells.

Angiogenesis assay

An angiogenesis assay (TCS Cellworks, England) was utilised to measure the stimulation of a 20% solution of stored blood with TCS supplied growth medium. Each well was incubated for ten days at which time the formed tubules were fixed with ethanol and immunostained. Control was compared to treatment with 2ng/ml of VEGF, suramin and 1 μg/ml of anti-VEGF antibody.

Primary antibody (mouse anti-human CD31) diluted 1 :4,000 in Blocking Buffer was added per well and incubated. Secondary antibody (goat anti-mouse IgG alkaline phosphatase conjugate) was diluted 1 :500 in blocking buffer. Wells were washed three times with Blocking Buffer and incubated prior to adding the secondary antibody that was also incubated at 37°C. Substrate was added and after 10 minutes tubules representing immature vessels became visible and substrate was removed. Each well was then washed three times with distilled water, the final wash was discarded and the plates left to air dry.

Analysis of the tubule lengths, used as a marker for angiogenesis stimulation, utilised the "AngioSys" image analysis system developed specifically for this angiogenesis assay. For every test well, which were blinded to the assessor at TCS Cellworks, four images were taken from as close to the centre of each quadrant from predetermined positions. The resultant tubule images were measured and compared. All statistical analyses were independently carried out using the Stat 100 programme from BIOSOFT Ltd. using ANOVA and Duncan's Multiple Comparison Test to measure differences between the test substances with control values. RESULTS

Clinical study of angiogenic factors in cancer patients

The results showed that the malignant group of patients [53.0 ng/ml (quartiles 38.2, 74.4)] had significantly lower plasma endostatin compared to controls [86.0 ng/ml (quartiles 44.9,131 P=0.01)]. The VEGF concentration of plasma [199.4 pg/ml (+/- 280.5)] in the malignant group was both significantly higher than that of the control group [78.9 pg/ml (+/- 64, P=0.01)]. Plasma endostatin and VEGF were not significantly correlated in the cancer (P=0.21) or control groups (P=0.31) suggesting they may be independent angiogenic factors. Platelet counts were significantly elevated in the cancer group (332.4 +/- 175.4) compared to the control group (230.2 +/- 76.3, P<0.05). Unexpectedly, control endostatin levels were not associated with platelet counts, but positively correlated with neutrophil counts (R= 0.42, P=0.05), which suggests neutrophils, already known to store VEGF, may also interact with circulating endostatin.

Study of stored blood products

Endostatin in packed red blood cells (2100 pg/ml +/- 810) became undetectable within one week of storage, during which time platelets increased bFGF levels by fourfold (4.4 +/- 1.9 vs. 17.4 +/- 4.5 pg/ml, P<0.001). Plasma from whole blood contained significantly more VEGF (73.6 pg/ml +/- 5.0) than leukodepleted packed cells (22.6 pg/ml +/- 1.2, P<0.01). However both fresh citrated whole blood and packed cells both accumulated VEGF during 15 days storage. Platelet pools had a high level of VEGF (308 pg/ml +/- 40). Buffy coat VEGF (2560 pg/ml +/- 404) content also significantly increased in concentration during storage.

SUMMARY

The accumulation of the angiogenic factor vascular endothelial growth factor is not matched by its antiangiogenic counterpart, endostatin. In addition, stored platelets significantly accumulate the other most studied angiogenic factor, basic fibroblast growth factor (bFGF). Stored blood significantly increases endothelial growth compared to fresh blood (P<0.05). A colorectal cancer cell line, CACO-2, increases proliferation upon treatment with stored blood supernatant compared to plasma of benign (P<0.05) or malignant patients. The proliferation of mature endothelial cells demonstrates that stored blood induces significantly more mitosis than benign patients serum (P<0.05). Endothelial proliferation and angiogenesis are stimulated by stored blood supernatant, an effect that is significantly reduced by anti-VEGF antibody (P<0.05). These data suggest that stored blood products stimulate both cancer cells and endothelial proliferation. This angiogenesis essential for tumour growth and metastases is inhibited by an anti-VEGF antibody.

DISCUSSION

Endostatin is an important endogenous agent that has been shown to inhibit endothelial proliferation. It is unclear currently how and where endostatin is synthesised though it is likely to be released by the proteolytic action of cancer invasion. We have previously shown that circulating endostatin levels are significantly reduced in colorectal cancer patients (unpublished data). Furthermore endostatin seems to be more concentrated in those cells within the buffy coat, including white blood cells. This finding supports the finding that endostatin levels are correlated with the neutrophils count of patients. The finding that endostatin levels are undetectable in buffy coat depleted blood after two weeks storage, subsequently increasing when the blood is stored to 35 days (data not shown), suggests the storage time of administered blood may be an important factor. This is perhaps explainable by the half-life of circulating free endostatin being short, but degradation of circulating cells may subsequently increase endostatin levels.

Elevated levels of bFGF have been linked to the switch to an angiogenic phenotype, which is thought essential for tumour growth. We found no significant change in bFGF levels after storage of various blood products, other than platelet pools that accumulate bFGF after six days.

We have shown that the circulating levels of bFGF, VEGF and endostatin are significantly different between cancer and control patients. Stored blood from the national blood bank stimulated mature endothelial cells significantly more than fresh blood from healthy controls. This result confirms the significance of the apparent accumulation of bioactive substances upon storage of blood. Furthermore there is a trend to increase proliferation of cancer cells and endothelium using the conditioned medium of cancer patients compared to control patients. However there is significantly more stimulation on addition of supernatant from stored blood reaching its expiry date.

These data suggest that in addition to any immunosuppressive effect blood transfusion may have, there is a positive proliferative effect on both colorectal tumour cells and endothelial cells at least in vitro. In addition the angiogenic stimulation effect seems to be contributed to by VEGF, which may be significantly inhibited by the addition of anti-VEGF antibody.

Claims

1. A method of modifying a blood product comprising altering the balance of angiogenic and anti-angiogenic factors therein, such that after storage or manipulation, said blood product has reduced angiogenic activity as compared to unmodified blood.

2. A method of modifying a blood product according to claim 1 , characterised in that said blood product has been stored or manipulated and that the level of an active angiogenic factor therein is depleted.

3. The method of Claim 2, wherein said active angiogenic factor is VEGF.

4. The method of Claim 2, wherein said active angiogenic factor is bFGF.

5. A method of modifying a stored or manipulated blood product according to either of claims 2 to 4, characterised by the depletion of two or more active angiogenic factors therein.

6. The method of claim 5, wherein said factors include VEGF and bFGF.

7. The method of any one of Claims 2 to 6, wherein the level of said one or more active angiogenic factors is depleted by means of affinity purification

8. The method of Claim 5, wherein said one or more angiogenic factors are depleted by means of immunoaffinity purification.

9. The method of Claim 6 wherein said said immunoaffinity purification is by means of one or more monoclonal antibodies or functional fragments thereof.

10. The method of any one of Claims 2 to 6, wherein the level of at least one active angiogenic factor is depleted by means of addition of an antagonist.

11. The method of Claim 10, wherein the antagonist binds to, and inactivates, said angiogenic factor.

12. The method of claim 11 , wherein the antagonist is a soluble receptor or functional fragment thereof.

13. The method of Claim 12 wherein the soluble receptor is an FGF receptor or VEGF receptor.

14. The method of Claim 11 , wherein the antagonist is an antibody or functional fragment thereof.

15. A method of modifying a blood product of any of the preceding claims, characterised by the addition of one or more anti-angiogenic factors.

16. The method of Claim 15, wherein the one or more anti-angiogenic factors added are endogenous to said blood product.

17. The method of either of claims 15 or 16, wherein the one or more anti-angiogenic factors are recombinant.

18. The method of any one of claims 15 to 17 wherein the one or more antiangiogenic factors are human factors.

19. The method of claims 18, wherein the one or more anti-angiogenic factors are modified human factors.

20. The method of either of claims 15 to 17, wherein the one or more anti-angiogenic factors are non-human factors.

21. The method of any of Claims 15 to 20, wherein one of said anti-angiogenic factors is endostatin.

22. A blood product prepared using the method of any of the preceding claims.

23. A blood product, characterised in that the level of an active angiogenic factor is depleted and/or the level of an anti-angiogenic factor is raised.

24. The blood product of claim 23, which further includes the feature(s) recited in one or more of claims 3 to 5.

25. The blood product of claim 23 or claim 24 which contains angiogenic factor bound to an antagonist.

26. The blood product of claim 25 wherein the antagonist is as defined in claim 12 or claim 13

27. The blood product of any of claims 23 to 26, characterised in that it comprises an anti-angiogenic factor distinguishable from nature-identical factors.

28. The blood product of claim 27 which further includes the feature(s) recited in one or more of claims 15 to 20.

29.Theblood product of any of claims 22 to 28, characterised in that it is whole blood.

30. The blood product of any of claims 22 to 28, characterised in that it is leukodepleted blood.

31. The blood product of any of claims 22 to 28, characterised in that it is essentially cell-free.

32. The blood product of any of claims 22 to 31 for use a medicament

33. The blood product of Claim 32 for use a medicament for the treatment of cancer.

34. The use of the blood product of any one of Claims 22 to 31 for the manufacture of a medicament for the treatment of cancer.

35. A method of treatment comprising administering to a patient the blood product of any one of Claims 22 to 31.

36. The method of claim 35, comprising a perioperative blood transfusion administered to a patient suffering from cancer.

37. A pharmaceutical composition comprising a blood product according to any one of claims 22 to 31.

38. A kit for the depletion of angiogenic factors from a blood product, comprising ligands capable of specific binding to one or more angiogenic factors and a means of binding and removing from solution complexes formed between said angiogenic factors and said ligands.

39. The kit of claim 38, wherein said ligands are antibodies or functional fragments thereof.

40. A sterile vessel containing the blood product of any of claims 22 to 31.

41.A vessel according to Claim 40, wherein the vessel is in the form of a bag.

42. A vessel according to either of Claims 40 or 41 , characterised in that it contains an anti-angiogenic factor.

43. A vessel according to either of Claims 40 or 41 , characterised in that it contains an angiogenic factor antagonist.

44. The vessel of either of Claims 42 or 43, characterised in that said anti-angiogenic factor or antagonist is immobilised to the inner surface of said vessel.