Hsp70 in Liquid Biopsies-A Tumor-Specific Biomarker for Detection and Response Monitoring in Cancer.

In contrast to normal cells, tumor cells of multiple entities overexpress the Heat shock protein 70 (Hsp70) not only in the cytosol, but also present it on their plasma membrane in a tumor-specific manner. Furthermore, membrane Hsp70-positive tumor cells actively release Hsp70 in small extracellular vesicles with biophysical characteristics of exosomes. Due to conformational changes of Hsp70 in a lipid environment, most commercially available antibodies fail to detect membrane-bound and vesicular Hsp70. To fill this gap and to assess the role of vesicular Hsp70 in circulation as a potential tumor biomarker, we established the novel complete (comp)Hsp70 sandwich ELISA, using two monoclonal antibodies (mAbs), that is able to recognize both free and lipid-associated Hsp70 on the cell surface of viable tumor cells and on small extracellular vesicles. The epitopes of the mAbs cmHsp70.1 (aa 451-461) and cmHsp70.2 (aa 614-623) that are conserved among different species reside in the substrate-binding domain of Hsp70 with measured affinities of 0.42 nM and 0.44 nM, respectively. Validation of the compHsp70 ELISA revealed a high intra- and inter-assay precision, linearity in a concentration range of 1.56 to 25 ng/mL, high recovery rates of spiked liposomal Hsp70 (>84%), comparable values between human serum and plasma samples and no interference by food intake or age of the donors. Hsp70 concentrations in the circulation of patients with glioblastoma, squamous cell or adeno non-small cell lung carcinoma (NSCLC) at diagnosis were significantly higher than those of healthy donors. Hsp70 concentrations dropped concomitantly with a decrease in viable tumor mass upon irradiation of patients with approximately 20 Gy (range 18-22.5 Gy) and after completion of radiotherapy (60-70 Gy). In summary, the compHsp70 ELISA presented herein provides a sensitive and reliable tool for measuring free and vesicular Hsp70 in liquid biopsies of tumor patients, levels of which can be used as a tumor-specific biomarker, for risk assessment (i.e., differentiation of grade III vs. IV adeno NSCLC) and monitoring of therapeutic outcomes.

First In-Human Medical Imaging with a PASylated 89Zr-Labeled Anti-HER2 Fab-Fragment in a Patient with Metastatic Breast Cancer.

PURPOSE: PASylation® offers the ability to systematically tune and optimize the pharmacokinetics of protein tracers for molecular imaging. Here we report the first clinical translation of a PASylated Fab fragment (89Zr∙Df-HER2-Fab-PAS200) for the molecular imaging of tumor-related HER2 expression. METHODS: A patient with HER2-positive metastatic breast cancer received 37 MBq of 89Zr∙Df-HER2-Fab-PAS200 at a total mass dose of 70 µg. PET/CT was carried out 6, 24, and 45 h after injection, followed by image analysis of biodistribution, normal organ uptake, and lesion targeting. RESULTS: Images show a biodistribution typical for protein tracers, characterized by a prominent blood pool 6 h p.i., which decreased over time. Lesions were detectable as early as 24 h p.i. 89Zr∙Df-HER2-Fab-PAS200 was tolerated well. CONCLUSION: This study demonstrates that a PASylated Fab tracer shows appropriate blood clearance to allow sensitive visualization of small tumor lesions in a clinical setting.

PASylation of IL-1 receptor antagonist (IL-1Ra) retains IL-1 blockade and extends its duration in mouse urate crystal-induced peritonitis.

Interleukin-1 (IL-1) is a key mediator of inflammation and immunity. Naturally-occurring IL-1 receptor antagonist (IL-1Ra) binds and blocks the IL-1 receptor-1 (IL-1R1), preventing signaling. Anakinra, a recombinant form of IL-1Ra, is used to treat a spectrum of inflammatory diseases. However, anakinra is rapidly cleared from the body and requires daily administration. To create a longer-lasting alternative, PASylated IL-1Ra (PAS-IL-1Ra) has been generated by in-frame fusion of a long, defined-length, N-terminal Pro/Ala/Ser (PAS) random-coil polypeptide with IL-1Ra. Here, we compared the efficacy of two PAS-IL-1Ra molecules, PAS600-IL-1Ra and PAS800-IL-1Ra (carrying 600 and 800 PAS residues, respectively), with that of anakinra in mice. PAS600-IL-1Ra displayed markedly extended blood plasma levels 3 days post-administration, whereas anakinra was undetectable after 24 h. We also studied PAS600-IL-1Ra and PAS800-IL-1Ra for efficacy in monosodium urate (MSU) crystal-induced peritonitis. 5 days post-administration, PAS800-IL-1Ra significantly reduced leukocyte influx and inflammatory markers in MSU-induced peritonitis, whereas equimolar anakinra administered 24 h before MSU challenge was ineffective. The 6-h pretreatment with equimolar anakinra or PAS800-IL-1Ra before MSU challenge similarly reduced inflammatory markers. In cultured A549 lung carcinoma cells, anakinra, PAS600-IL-1Ra, and PAS800-IL-Ra reduced IL-1α-induced IL-6 and IL-8 levels with comparable potency. In human peripheral blood mononuclear cells, these molecules suppressed Candida albicans-induced production of the cancer-promoting cytokine IL-22. Surface plasmon resonance analyses revealed significant binding between PAS-IL-1Ra and IL-1R1, although with a slightly lower affinity than anakinra. These results validate PAS-IL-1Ra as an active IL-1 antagonist with marked in vivo potency and a significantly extended half-life compared with anakinra.

PASylated interferon α efficiently suppresses hepatitis B virus and induces anti-HBs seroconversion in HBV-transgenic mice.

Interferon α (IFNα) so far is the only therapeutic option for chronic hepatitis B virus (HBV) infection that can lead to virus clearance. Unfortunately, its application is limited by side effects and response rates are low. The aim of this study was to generate a novel long-acting IFNα with the help of PASylation technology that adds a polypeptide comprising Proline, Alanine and Serine (PAS) to increase plasma half-life. Following evaluation of four selected recombinant murine IFNα (mIFNα) subtypes in cell culture, the most active subtype, mIFNα11, was fused with a 600 amino acid PAS chain. The activity of PAS-mIFNα was assessed by interferon bioassay and further evaluated for induction of interferon-stimulated genes (ISG) and antiviral efficacy in cell culture as well as in HBV-transgenic mice. PAS-mIFNα induced expression of ISG comparable to unmodified mIFNα and, likewise, evoked dose-dependent reduction of HBV replication in vitro. In vivo, PAS-mIFNα led to pronounced suppression of HBV replication without detectable liver damage whereas conventional mIFNα treatment only had a modest antiviral effect. Importantly, all PAS-mIFNα treated mice showed an anti-HBs antibody response, lost HBsAg and achieved seroconversion after three weeks. PASylated IFNα showed a profoundly increased antiviral effect in vivo compared to the non-modified version without toxicity, providing proof-of-concept that an improved IFNα can achieve higher rates of HBV antiviral and immune control.

Regeneration After Radiation- and Immune-Mediated Tissue Injury Is Not Enhanced by Type III Interferon Signaling.

PURPOSE: Type I interferon (IFN-I) and interleukin (IL)-22 modulate regeneration of the thymus and intestinal epithelial cells (IECs) after cytotoxic stress such as irradiation. Radiation-induced damage to thymic tissues and IECs is a crucial aspect during the pathogenesis of inadequate immune reconstitution and acute graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) with myeloablative total body irradiation (TBI), respectively. IL-22 and IFN-I reduce the severity of acute GVHD after allo-HSCT with myeloablative TBI. However, the role of biologically related type III interferon (IFN-III), also known as interferon lambda (IFN-λ) or IL-28, in this context is unclear. We therefore studied the role of the IFN-III pathway in thymic regeneration and GVHD after TBI and allo-HSCT. METHODS AND MATERIALS: Cohoused wild-type (WT) and IFN-III receptor-deficient (IL-28 receptor alpha subunit-deficient/IL-28Ra-/-) mice were analyzed in models of TBI-induced thymus damage and a model of GVHD after allo-HSCT with myeloablative TBI. PASylated IFN-III (PASylated IL-28A, XL-protein GmbH) was generated to prolong the plasma half-life of IFN-III. Pharmacologic activity and the effects of PASylated IL-28A on radiation-induced thymus damage and the course of GVHD after allo-HSCT with myeloablative TBI were tested. RESULTS: The course and severity of GVHD after myeloablative TBI and allo-HSCT in IL-28Ra-/- mice was comparable to those in WT mice. Activation of the IFN-III pathway by PASylated IL-28A did not significantly modulate GVHD after allo-HSCT with TBI. Furthermore, IL28Ra-/- mice and WT mice showed similar thymus regeneration after radiation, which could also not be significantly modulated by IFN-III receptor engagement using PASylated IL-28A. CONCLUSIONS: We analyzed the role of IFN-III signaling during radiation-mediated acute tissue injury. Despite molecular and biologic homologies with IFN-I and IL-22, IFN-III signaling did not improve thymus regeneration after radiation or the course of GVHD after myeloablative TBI and allo-HSCT.

PASylated Coversin, a C5-Specific Complement Inhibitor with Extended Pharmacokinetics, Shows Enhanced Anti-Hemolytic Activity in Vitro.

The Ornithodoros moubata Complement Inhibitor (OmCI) binds complement component 5 (C5) with high affinity and, thus, selectively prevents proteolytic activation of the terminal lytic complement pathway. A recombinant version of OmCI (also known as Coversin and rEV576) has proven efficacious in several animal models of complement-mediated diseases and successfully completed a phase Ia clinical trial. Coversin is a small 17 kDa lipocalin protein which has a very short plasma half-life if not bound to C5; therefore, the drug requires frequent dosing. We have improved the pharmacokinetics of Coversin by N-terminal translational conjugation with a 600 residue polypeptide composed of Pro, Ala, and Ser (PAS) residues. To this end, PAS-Coversin as well as the unmodified Coversin were functionally expressed in the cytoplasm of E. coli and purified to homogeneity. Both versions showed identical affinity to human C5, as determined by surface plasmon resonance measurements, and revealed similar complement inhibitory activity, as measured in ELISAs with human serum. In line with the PEG-like biophysical properties, PASylation dramatically prolonged the plasma half-life of uncomplexed Coversin by a factor ≥50 in mice. In a clinically relevant in vitro model of the complement-mediated disease paroxysmal nocturnal hemoglobinuria (PNH) both versions of Coversin effectively reduced erythrocyte lysis. Unexpectedly, while the IC50 values were comparable, PAS-Coversin reached a substantially lower plateau of residual lysis at saturating inhibitor concentrations. Taken together, our data demonstrate two clinically relevant improvements of PASylated Coversin: markedly increased plasma half-life and considerably reduced background hemolysis of erythrocytes with PNH-induced phenotype.

Treatment of diet-induced lipodystrophic C57BL/6J mice with long-acting PASylated leptin normalises insulin sensitivity and hepatic steatosis by promoting lipid utilisation.

AIMS/HYPOTHESIS: Recombinant leptin offers a viable treatment for lipodystrophy (LD) syndromes. However, due to its short plasma half-life, leptin replacement therapy requires at least daily subcutaneous (s.c.) injections. Here, we optimised this treatment strategy in LD mice by using a novel leptin version with extended plasma half-life using PASylation technology. METHODS: A long-acting leptin version was prepared by genetic fusion with a 600 residue polypeptide made of Pro, Ala and Ser (PASylation), which enlarges the hydrodynamic volume and, thus, retards renal filtration, allowing less frequent injection. LD was induced in C57BL/6J mice by feeding a diet supplemented with conjugated linoleic acid (CLA). Chronic and acute effects of leptin treatment were assessed by evaluating plasma insulin levels, insulin tolerance, histological liver sections, energy expenditure, energy intake and body composition. RESULTS: In a cohort of female mice, 4 nmol PAS-leptin (applied via four s.c. injections every 3 days) successfully alleviated the CLA-induced LD phenotype, which was characterised by hyperinsulinaemia, insulin intolerance and hepatosteatosis. The same injection regimen had no measurable effect when unmodified recombinant leptin was administered at an equivalent dose. In a cohort of LD males, a single s.c. injection of PAS-leptin did not affect energy expenditure but inhibited food intake and promoted a shift in fuel selection towards preferential fat oxidation, which mechanistically substantiates the metabolic improvements. CONCLUSIONS/INTERPRETATION: The excellent pharmacological properties render PASylated leptin an agent of choice for refining both animal studies and therapeutic strategies in the context of LD syndromes and beyond.

Long-Acting PASylated Leptin Ameliorates Obesity by Promoting Satiety and Preventing Hypometabolism in Leptin-Deficient Lep(ob/ob) Mice.

Body weight loss of Lep(ob/ob) mice in response to leptin is larger than expected from the reduction in energy intake alone, suggesting a thermogenic action of unknown magnitude. We exploited the superior pharmacological properties of a novel long-acting leptin prepared via PASylation to study the contribution of its anorexigenic and thermogenic effects. PASylation, the genetic fusion of leptin with a conformationally disordered polypeptide comprising 600 Pro/Ala/Ser (PAS) residues, provides a superior way to increase the hydrodynamic volume of the fusion protein, thus retarding kidney filtration and extending plasma half-life. Here a single PAS(600)-leptin injection (300 pmol/g) resulted in a maximal weight reduction of 21% 6 days after application. The negative energy balance of 300 kJ/(4 d) was driven by a decrease in energy intake, whereas energy expenditure remained stable. Mice that were food restricted to the same extent showed an energy deficit of only 220 kJ/(4 d) owing to recurring torpor bouts. Therefore, the anorexigenic effect of PAS(600)-leptin contributes 75% to weight loss, whereas the thermogenic action accounts for 25% by preventing hypometabolism. In a second experiment, just four injections of PAS(600)-leptin (100 pmol/g) administered in 5- to 6-day intervals rectified the Lep(ob/ob) phenotype. In total, 16 nmol of PAS(600)-leptin per mouse triggered a weight loss of 43% within 20 days and normalized hypothermia and glucose homeostasis as well as hepatic steatosis. The beneficial properties of PAS(600)-leptin are substantiated by a comparison with previous studies in which approximately 400 nmol (∼25-fold) unmodified leptin was mandatory to achieve similar improvements.

PASylation of Murine Leptin Leads to Extended Plasma Half-Life and Enhanced in Vivo Efficacy.

Leptin plays a central role in the control of energy homeostasis and appetite and, thus, has attracted attention for therapeutic approaches in spite of its limited pharmacological activity owing to the very short circulation in the body. To improve drug delivery and prolong plasma half-life, we have fused murine leptin with Pro/Ala/Ser (PAS) polypeptides of up to 600 residues, which adopt random coil conformation with expanded hydrodynamic volume in solution and, consequently, retard kidney filtration in a similar manner as polyethylene glycol (PEG). Relative to unmodified leptin, size exclusion chromatography and dynamic light scattering revealed an approximately 21-fold increase in apparent size and a much larger molecular diameter of around 18 nm for PAS(600)-leptin. High receptor-binding activity for all PASylated leptin versions was confirmed in BIAcore measurements and cell-based dual-luciferase assays. Pharmacokinetic studies in mice revealed a much extended plasma half-life after ip injection, from 26 min for the unmodified leptin to 19.6 h for the PAS(600) fusion. In vivo activity was investigated after single ip injection of equimolar doses of each leptin version. Strongly increased and prolonged hypothalamic STAT3 phosphorylation was detected for PAS(600)-leptin. Also, a reduction in daily food intake by up to 60% as well as loss in body weight of >10% lasting for >5 days was observed, whereas unmodified leptin was merely effective for 1 day. Notably, application of a PASylated superactive mouse leptin antagonist (SMLA) led to the opposite effects. Thus, PASylated leptin not only provides a promising reagent to study its physiological role in vivo but also may offer a superior drug candidate for clinical therapy.

High contrast tumor imaging with radio-labeled antibody Fab fragments tailored for optimized pharmacokinetics via PASylation.

Although antigen-binding fragments (Fabs) of antibodies constitute established tracers for in vivo radiodiagnostics, their functionality is hampered by a very short circulation half-life. PASylation, the genetic fusion with a long, conformationally disordered amino acid chain comprising Pro, Ala and Ser, provides a convenient way to expand protein size and, consequently, retard renal filtration. Humanized αHER2 and αCD20 Fabs were systematically fused with 100 to 600 PAS residues and produced in E. coli. Cytofluorimetric titration analysis on tumor cell lines confirmed that antigen-binding activities of the parental antibodies were retained. The radio-iodinated PASylated Fabs were studied by positron emission tomography (PET) imaging and biodistribution analysis in mouse tumor xenograft models. While the unmodified αHER2 and αCD20 Fabs showed weak tumor uptake (0.8% and 0.2% ID/g, respectively; 24 h p.i.) tumor-associated radioactivity was boosted with increasing PAS length (up to 9 and 26-fold, respectively), approaching an optimum for Fab-PAS400. Remarkably, 6- and 5-fold higher tumor-to-blood ratios compared with the unmodified Fabs were measured in the biodistribution analysis (48 h p.i.) for αHER2 Fab-PAS100 and Fab-PAS200, respectively. These findings were confirmed by PET studies, showing high imaging contrast in line with tumor-to-blood ratios of 12.2 and 5.7 (24 h p.i.) for αHER2 Fab-PAS100 and Fab-PAS200. Even stronger tumor signals were obtained with the corresponding αCD20 Fabs, both in PET imaging and biodistribution analysis, with an uptake of 2.8% ID/g for Fab-PAS100 vs. 0.24% ID/g for the unmodified Fab. Hence, by engineering Fabs via PASylation, plasma half-life can be tailored to significantly improve tracer uptake and tumor contrast, thus optimally matching reagent/target interactions.

Enhanced in vivo efficacy of a type I interferon superagonist with extended plasma half-life in a mouse model of multiple sclerosis.

IFNß is a common therapeutic option to treat multiple sclerosis. It is unique among the family of type I IFNs in that it binds to the interferon receptors with high affinity, conferring exceptional biological properties. We have previously reported the generation of an interferon superagonist (dubbed YNSα8) that is built on the backbone of a low affinity IFNα but modified to exhibit higher receptor affinity than even for IFNß. Here, YNSα8 was fused with a 600-residue hydrophilic, unstructured N-terminal polypeptide chain comprising proline, alanine, and serine (PAS) to prolong its plasma half-life via "PASylation." PAS-YNSα8 exhibited a 10-fold increased half-life in both pharmacodynamic and pharmacokinetic assays in a transgenic mouse model harboring the human receptors, notably without any detectable loss in biological potency or bioavailability. This long-lived superagonist conferred significantly improved protection from MOG35-55-induced experimental autoimmune encephalomyelitis compared with IFNß, despite being injected with a 4-fold less frequency and at an overall 16-fold lower dosage. These data were corroborated by FACS measurements showing a decrease of CD11b(+)/CD45(hi) myeloid lineage cells detectable in the CNS, as well as a decrease in IBA(+) cells in spinal cord sections determined by immunohistochemistry for PAS-YNSα8-treated animals. Importantly, PAS-YNSα8 did not induce antibodies upon repeated administration, and its biological efficacy remained unchanged after 21 days of treatment. A striking correlation between increased levels of CD274 (PD-L1) transcripts from spleen-derived CD4(+) cells and improved clinical response to autoimmune encephalomyelitis was observed, indicating that, at least in this mouse model of multiple sclerosis, CD274 may serve as a biomarker to predict the effectiveness of IFN therapy to treat this complex disease.

PASylation: a biological alternative to PEGylation for extending the plasma half-life of pharmaceutically active proteins.

A major limitation of biopharmaceutical proteins is their fast clearance from circulation via kidney filtration, which strongly hampers efficacy both in animal studies and in human therapy. We have developed conformationally disordered polypeptide chains with expanded hydrodynamic volume comprising the small residues Pro, Ala and Ser (PAS). PAS sequences are hydrophilic, uncharged biological polymers with biophysical properties very similar to poly-ethylene glycol (PEG), whose chemical conjugation to drugs is an established method for plasma half-life extension. In contrast, PAS polypeptides offer fusion to a therapeutic protein on the genetic level, permitting Escherichia coli production of fully active proteins and obviating in vitro coupling or modification steps. Furthermore, they are biodegradable, thus avoiding organ accumulation, while showing stability in serum and lacking toxicity or immunogenicity in mice. We demonstrate that PASylation bestows typical biologics, such as interferon, growth hormone or Fab fragments, with considerably prolonged circulation and boosts bioactivity in vivo.

High molecular mass radioimmunoconjugates are promising for intraperitoneal α-emitter immunotherapy due to prolonged retention in the peritoneum.

INTRODUCTION: Therapeutic efficacy of intraperitoneal radioimmunotherapy is dependent on the time of retention of the radioimmunoconjugates within the peritoneal cavity. Therefore, the aim of this study was to investigate intraperitoneal retention of Fab, IgG and IgM radioimmunoconjugates. METHODS: Female Balb/c mice were injected with 213Bi- or 111In-labeled IgM, IgG and recombinant Fab conjugates intraperitoneally or intravenously. At different time points after injection, whole body distribution of radionuclides was imaged using a gamma camera. Distribution of radionuclides in selected organs was determined via γ-counting after sacrifice. Biological half-lives of the conjugates were calculated from whole body activities. RESULTS: After i.p. injection 213Bi-Fab rapidly accumulated in the kidneys indicative of glomerular filtration and reabsorption. Accumulation of 213Bi-IgG in the kidneys was significantly lower. 213Bi-IgM showed a striking accumulation in the liver 180 min after i.p. injection. 111In-IgG persisted in the circulation up to 72 h both after i.p. and i.v. injection. 111In-IgM showed a continuous accumulation in the liver. Moreover, 111In-IgM was significantly higher 24 h after i.v. injection than i.p. injection both in liver and spleen. These differences could be confirmed via scintigraphy. After injection of 111In-IgG differences in scintigraphic images between i.v. and i.p. were clearly visible only at 3 h. Biological half lives were 24 h, 45 h and 165 h for 111In-IgM, 111In-Fab and 111In-IgG, respectively. CONCLUSIONS: Retention of radioimmunoconjugates in the peritoneal cavity positively correlates with the molecular mass of the antibody. Therefore, IgM radioimmunoconjugates should be preferably used in radioimmunotherapy of free floating tumor cells and small tumor cell clusters in the ascites of the peritoneal cavity.

Functional humanization of an anti-CD16 Fab fragment: obstacles of switching from murine {lambda} to human {lambda} or {kappa} light chains.

An alphaCD30xalphaCD16 bispecific monoclonal antibody (MAb) was previously shown to induce remission of Hodgkin's disease refractory to chemo- and radiotherapy through specific activation of natural killer (NK) cells, but the appearance of a human anti-mouse antibody (HAMA) response prevented its use for prolonged therapy. Here, we describe an effort to humanize the Fab arm directed against FcgammaRIII (CD16), which-in context with the previously humanized CD30 Fab fragment-provides the necessary component for the design of a clinically useful bispecific antibody. Thus, the CDRs of the anti-CD16 mouse IgG1/lambda MAb A9 were grafted onto human Ig sequences. In a first attempt, the murine V(lambda) domain was converted to a humanized lambda chain, which led, however, to complete loss of antigen-binding activity and extremely poor folding efficiency upon periplasmic expression in Escherichia coli. Hence, its CDRs were transplanted onto a human kappa light chain in a second attempt, which resulted in a functional recombinant Fab fragment, yet with 100-fold decreased antigen affinity. In the next step, an in vitro affinity maturation was performed, wherein random mutations were introduced into the humanized V(H) and V(kappa) domains through error-prone PCR, followed by a filter sandwich colony screening assay for increased binding activity towards the bacterially produced extracellular CD16 fragment. Finally, an optimized Fab fragment was obtained, which carries nine additional amino acid exchanges and exhibits an affinity that is within a factor of 2 identical to that of the original murine A9 Fab fragment. The resulting humanized Fab fragment was fully functional with respect to binding of the recombinant CD16 antigen in enzyme-linked immunosorbent assay and in cytofluorimetry with CD16-positive granulocytes, thus providing a promising starting point for the preparation of a fully human bispecific antibody that permits the therapeutic recruitment of NK cells.

Fusion of a recombinant antibody fragment with a homo-amino-acid polymer: effects on biophysical properties and prolonged plasma half-life.

Chemical conjugation of small recombinant proteins with polyethylene glycol (PEG) is an established strategy to extend their typically short circulation times to a therapeutically useful range. We have investigated the production of a genetic fusion with a glycine-rich homo-amino-acid polymer (HAP) as an alternative way to attach a solvated random chain with large hydrodynamic volume. The anti-HER2 Fab fragment 4D5 was used as a model system and fused with either 100 or 200 residue polymers of the repetitive sequence (Gly(4)Ser)(n) to its light chain. Both fusion proteins were successfully produced in the periplasm of Escherichia coli and obtained as homogeneous preparations after two-step affinity chromatography via the His(6) tag fused to the heavy chain and the Strep-tag II fused to the extended light chain. Both modified Fab fragments showed binding activity towards the HER2 antigen indistinguishable from the conventional recombinant Fab fragment. When compared with the unfused Fab fragment, a significantly increased hydrodynamic volume, by ca. 120%, was observed during gel filtration for the 200 residue HAP fusion protein and, to a lesser extent, in the case of the 100 residue HAP. Difference CD measurements revealed a characteristic random coil spectrum for the 100 and 200 residue HAP fusion moieties. Finally, pharmacokinetic experiments were carried out in mice after radioiodination of the recombinant Fab fragments. Although the 100 residue HAP fusion showed a behavior very similar to the unfused Fab fragment, with a terminal plasma half-life of ca. 2 h, the 200 residue HAPylated Fab fragment gave rise to a significantly prolonged half-life of ca. 6 h. While this moderate effect may so far be most beneficial for specialized medical applications, such as in vivo imaging, the genetic engineering of optimized HAP sequences should yield pharmacokinetic properties similar to PEGylation, yet without necessitating in vitro modification steps.

Functional humanization of an anti-CD30 Fab fragment for the immunotherapy of Hodgkin's lymphoma using an in vitro evolution approach.

CD30, the so-called Reed-Sternberg antigen, constitutes a promising cell-specific target for the treatment of Hodgkin's lymphoma. Starting from the previously characterized cognate HRS3 mouse monoclonal antibody, the bacterially produced functional Fab fragment was humanized by grafting the CDRs from the mouse antibody framework on to human immunoglobulin consensus sequences. This procedure led to a 10-fold decreased antigen affinity, which surprisingly was found to be mainly due to the VH domain. To improve the antigen-binding activity, an in vitro evolution strategy was employed, wherein random mutations were introduced into the humanized VH domain by means of error-prone PCR, followed by a filter sandwich Escherichia coli colony screening assay for functional Fab fragments using a recombinant extracellular domain of the CD30 antigen. After three cycles of in vitro affinity maturation, the optimized Fab fragment huHRS3-VH-EP3/1 was identified, which carried four exchanged residues within or close to the VH CDRs and had an affinity that was almost identical with that of the murine HRS3 Fab fragment. The resulting humanized Fab fragment was fully functional with respect to CD30 binding both in ELISA with the recombinant antigen and in FACS experiments with CD30-positive L540CY cells. In the light of the previously successful clinical application of an alphaCD30 x alphaCD16 bispecific mouse quadroma antibody derived from HRS3, the humanized Fab fragment comprises an important step towards the construction of a fully recombinant therapeutic agent. The combination of random mutagenesis and colony filter screening assay that was successfully applied here should be generally useful as a method for the rapid functional optimization of humanized antibody fragments.