TWI676481B - Induction of IL-12 using immunotherapy - Google Patents

Abstract

本發明係關於促進患者之IL-12誘導之組成物及方法。該組成物包含向患有諸如癌症之疾病之患者投予的活化的同種異體細胞。投予該組成物使患者之免疫反應偏向Th1環境且在患者血漿中產生可偵測含量之IL-12而無任何IL-12相關毒性。 The present invention relates to compositions and methods for promoting IL-12 induction in patients. The composition comprises activated allogeneic cells administered to a patient suffering from a disease such as cancer. Administration of this composition biases the patient's immune response towards the Th1 environment and produces detectable levels of IL-12 in the patient's plasma without any IL-12-related toxicity.

Description

使用免疫療法誘導IL-12 Induction of IL-12 using immunotherapy

本發明係關於使用免疫細胞之療法。更詳言之，本發明係關於促進患者之IL-12產生之免疫細胞療法。 The present invention relates to therapy using immune cells. More specifically, the present invention relates to immune cell therapies that promote IL-12 production in patients.

已知最精確、最強力及最安全之疾病預防及治療機制為天然『消毒性(sterilizing)』免疫反應，其組合先天性免疫性與繼承性免疫性兩者之要素以使身體在無醫學介入下免於多種外來病原體之憂慮。免疫系統係設計來『記住』所清除之外來抗原以在再感染時快速產生免疫反應。免疫系統，甚至癌症患者之免疫系統亦可識別諸如見於病毒及細菌上之外來抗原且對其產生足以將其完全破壞並使其自身體消除之反應。此消毒性免疫反應之兇猛性及特異性可以以下方式證明：未受適當抑制之免疫系統可完全破壞大型移植器官，諸如腎、肝或心臟，同時不傷害自身組織。若針對外來抗原之此免疫性之破壞作用可再針對因患者免疫反應不足而逃脫之腫瘤及/或其他抗原，則此作用將為有益的。 The most accurate, powerful, and safest disease prevention and treatment mechanism is known as the natural "sterilizing" immune response, which combines the elements of innate immunity and inherited immunity to enable the body without medical intervention Free from the worry of many foreign pathogens. The immune system is designed to "remember" the foreign antigens that are cleared to rapidly generate an immune response during reinfection. The immune system, and even the immune system of cancer patients, can recognize foreign antigens such as those found on viruses and bacteria and produce a response sufficient to completely destroy them and eliminate themselves. The fierceness and specificity of this sterilizing immune response can be demonstrated in the following way: The immune system, which is not properly suppressed, can completely destroy large transplanted organs, such as kidney, liver or heart, without harming its own tissues. This effect would be beneficial if the damaging effect of this immunity on foreign antigens could be directed against tumors and / or other antigens that escaped due to the patient's insufficient immune response.

免疫療法致力於開發用以利用、引導及控制針對多種感染性及非感染性疾病(包含癌症)之免疫反應之方法。治療疫苗為一種設計來教化免疫系統之免疫療法之類型。在存在癌症之患者中，疫苗經設計，因此患者之免疫系統將腫瘤細胞識別為外來物。若免疫系統將腫瘤識別為外來病原體，則理論上可引發免疫反應，其可使免疫細胞破壞 大腫瘤且找出並破壞轉移性腫瘤細胞，而無論其存在於身體中何處。在成功免疫療法之後，免疫系統『記住』所消除外來細胞之能力將使免疫系統能夠在無其他治療下消除任何複現之癌細胞，非常像免疫系統防止機會性感染。 Immunotherapy is dedicated to the development of methods to utilize, direct, and control immune responses to a variety of infectious and non-infectious diseases, including cancer. A therapeutic vaccine is a type of immunotherapy designed to educate the immune system. In patients with cancer, the vaccine is designed so that the patient's immune system recognizes tumor cells as foreign objects. If the immune system recognizes a tumor as a foreign pathogen, it can theoretically trigger an immune response, which can destroy immune cells Large tumors and find and destroy metastatic tumor cells wherever they exist in the body. After successful immunotherapy, the ability of the immune system to "remember" the elimination of foreign cells will enable the immune system to eliminate any recurring cancer cells without other treatments, much like the immune system prevents opportunistic infections.

個體免疫系統對疾病或疾病生物體之反應可為Th1反應或Th2反應。在Th1反應中，CD4+ T細胞變得向Th1細胞極化，且反之，在Th2反應中，CD4+ T細胞變得向Th2細胞極化。此日益盛行之分類方法稱為Th1/Th2平衡。Th1細胞促進細胞介導之免疫性，而Th2細胞誘導體液免疫性。 細胞免疫性(Th1)引導自然殺手細胞(NK)、T細胞及巨噬細胞攻擊感染部位之異常細胞及微生物。體液免疫性(Th2)造成用於中和外來侵入物之抗體的產生。一般而言，CD4+ T細胞之Th2極化已在大多數人類及動物癌症研究中顯示與癌症進展相關，而Th1極化與腫瘤消退及抗腫瘤免疫性相關。 The response of an individual's immune system to a disease or disease organism can be a Th1 response or a Th2 response. In the Th1 response, CD4 + T cells become polarized toward Th1 cells, and conversely, in the Th2 response, CD4 + T cells become polarized toward Th2 cells. This increasingly popular classification method is called Th1 / Th2 balance. Th1 cells promote cell-mediated immunity, while Th2 cells induce humoral immunity. Cell immunity (Th1) guides natural killer cells (NK), T cells, and macrophages to attack abnormal cells and microorganisms at the site of infection. Humoral immunity (Th2) results in the production of antibodies used to neutralize foreign invaders. In general, Th2 polarization of CD4 + T cells has been shown to be associated with cancer progression in most human and animal cancer studies, while Th1 polarization is associated with tumor regression and anti-tumor immunity.

個體之免疫反應(Th1/Th2平衡)可經由個體之細胞激素之平衡加以評估。細胞激素為小細胞信號傳導蛋白質分子。術語細胞激素用作多樣的組之可溶性蛋白質及肽之上位名稱，該等蛋白質及肽通常在奈莫耳至披(pico)莫耳濃度下充當調節劑且其在正常或病理學條件下調節個體細胞及組織之功能活性。此等蛋白質亦直接介導細胞之間的相互作用且調節在細胞外環境中進行之過程。介白素(Interleukin)為一組涉及免疫調節中之細胞激素且可由免疫系統中之多種細胞合成。存在許多介白素，諸如IL-2、 IL-4、IL-10及IL-12，且此等介白素各自在免疫系統內具有特定作用。 An individual's immune response (Th1 / Th2 balance) can be assessed by the individual's cytokine balance. Cytokines are small cell signaling protein molecules. The term cytokine is used as a superior name for a diverse group of soluble proteins and peptides, which generally act as regulators at concentrations of nanomole to picomole and which regulate individuals under normal or pathological conditions Functional activity of cells and tissues. These proteins also directly mediate interactions between cells and regulate processes that take place in the extracellular environment. Interleukin is a group of cytokines involved in immune regulation and can be synthesized by a variety of cells in the immune system. There are many interleukins, such as IL-2, IL-4, IL-10, and IL-12, and these interleukins each have a specific role in the immune system.

Th1細胞產生涉及於發炎反應中之1型細胞激素。1型細胞激素包含例如IL-2、IL-12、IL-15、IFN-γ、TNF-α、TNF-β、GM-CSF及C-C趨化素(chemokine)。Th2細胞產生涉及於體液免疫反應中之2型細胞激素。2型細胞激素包含例如IL-4、IL-5、IL-6、IL-10、IL-13及TGF-β。Th1及Th2免疫反應為具有相反調節性，以致1型反應增加會下調2型反應且2型反應增加會下調1型反應。 Th1 cells produce type 1 cytokines involved in the inflammatory response. Type 1 cytokines include, for example, IL-2, IL-12, IL-15, IFN-γ, TNF-α, TNF-β, GM-CSF, and C-C chemokine. Th2 cells produce type 2 cytokines involved in humoral immune responses. Type 2 cytokines include, for example, IL-4, IL-5, IL-6, IL-10, IL-13, and TGF-β. Th1 and Th2 immune responses have opposite regulatory effects, so that an increase in type 1 response will down-regulate a type 2 response and an increase in type 2 response will down-regulate a type 1 response.

IL-12為一種由p35及p40次單元構成之異二聚體。其主要由抗原呈現細胞(APC)產生。IL-12亦可由單核球及巨噬細胞、樹突細胞及B細胞產生。IL-12對T細胞及自然殺手細胞施加免疫調節作用。已知內源性IL-12涉及於產生最佳Th1反應且可在針對細胞內病原體之細胞介導之免疫性中起重要作用。 IL-12 is a heterodimer consisting of p35 and p40 subunits. It is mainly produced by antigen-presenting cells (APC). IL-12 can also be produced by monocytes and macrophages, dendritic cells and B cells. IL-12 exerts immunomodulatory effects on T cells and natural killer cells. Endogenous IL-12 is known to be involved in generating the optimal Th1 response and can play an important role in cell-mediated immunity against intracellular pathogens.

因為IL-12調節免疫系統之重要組分且已在實驗室及動物研究中證明具有顯著抗腫瘤作用，所以其已成為大量研究之主題。IL-12已例如牽涉於抑制人類肺腺癌生長及急性骨髓性白血病。然而，在治療方案中使用外源性IL-12已受限於在人類中具有高毒性。 Because IL-12 regulates an important component of the immune system and has been shown to have significant antitumor effects in laboratory and animal studies, it has become the subject of much research. IL-12 has been implicated, for example, in inhibiting human lung adenocarcinoma growth and acute myeloid leukemia. However, the use of exogenous IL-12 in treatment regimens has been limited to high toxicity in humans.

本發明係關於在患者血漿中產生可偵測含量之IL-12之組成物及方法。本發明包含一種組成物，其在向患者投予時可使得在該患者血漿中產生可偵測含量之內源性 IL-12，而無任何顯著毒性。驚人地可在癌症患者中偵測到內源性IL-12。組成物較佳包含同種異體活化的T細胞。T細胞不能夠產生IL-12，因此向患者投予之T細胞組成物引發由患者之自身APC產生IL-12。 The present invention relates to compositions and methods for producing detectable levels of IL-12 in the plasma of patients. The present invention comprises a composition which, when administered to a patient, causes endogenous production of a detectable content in the patient's plasma IL-12 without any significant toxicity. Surprisingly, endogenous IL-12 can be detected in cancer patients. The composition preferably comprises allo-activated T cells. T cells are not capable of producing IL-12, so the T cell composition administered to a patient triggers the production of IL-12 by the patient's own APC.

本發明亦包含誘導在患者體內由患者自身免疫系統產生內源性IL-12之方法。方法包括投予同種異體物質，較佳同種異體活化的T細胞之組成物。組成物可以單劑或多劑形式投予。較佳地，以頻繁之低劑量投予同種異體活化的T細胞。同種異體細胞可藉由皮內、靜脈內或病灶內途徑投予。較佳地，頻率不小於每3天1次。當投予此等組成物時，患者自身免疫系統可經誘導以在血漿中產生可偵測含量之內源性IL-12，甚至在癌症患者中亦如此。一般而言，因為腫瘤可抑制IL-12表現，所以IL-12不見於癌症患者中。驚人的是，本文所述之方法可克服此抑制且創建足以長時期(例如數月或甚至1年)在血漿中誘導IL-12表現之環境。此外，血漿中存在內源性IL-12不會在患者中導致如投予外源性IL-12作為醫藥品(medicant)所產生之顯著毒性。 The invention also includes a method for inducing endogenous IL-12 production by a patient's own immune system in a patient. The method includes administering an allogeneic substance, preferably a composition of allogeneic activated T cells. The composition may be administered in single or multiple doses. Preferably, allogeneic activated T cells are administered at frequent low doses. Allogeneic cells can be administered by intradermal, intravenous or intralesional routes. Preferably, the frequency is not less than once every 3 days. When administered to these compositions, the patient's own immune system can be induced to produce detectable levels of endogenous IL-12 in plasma, even in cancer patients. In general, IL-12 is not found in cancer patients because tumors can inhibit the expression of IL-12. Surprisingly, the methods described herein can overcome this inhibition and create an environment sufficient to induce IL-12 expression in plasma for a long period of time (e.g., months or even 1 year). In addition, the presence of endogenous IL-12 in plasma does not cause significant toxicity in patients such as administration of exogenous IL-12 as a medicant.

內源性IL-12意謂IL-12在患者體內由患者自身免疫系統合成。詳言之，IL-12可由患者之抗原呈現細胞(APC)合成。APC可包含單核球及巨噬細胞、樹突細胞及B細胞。 外源性IL-12意謂IL-12不由患者自身免疫系統合成。外源性IL-12包含自另一個體分離之IL-12及/或純化之IL-12，或藉由包含IL-12之基因之DNA構築體表現的IL-12。 Endogenous IL-12 means that IL-12 is synthesized by the patient's own immune system in the patient. In particular, IL-12 can be synthesized by a patient's antigen-presenting cells (APC). APCs can include monocytes and macrophages, dendritic cells and B cells. Exogenous IL-12 means that IL-12 is not synthesized by the patient's own immune system. Exogenous IL-12 includes IL-12 isolated and / or purified IL-12 from another body, or IL-12 expressed by a DNA construct containing a gene of IL-12.

有利的是，在患者體內全身性產生內源性IL-12對患者產生最小毒性或不對患者產生毒性。患者可經歷短暫症狀，諸如短暫流感樣症狀。一般而言，當已向患者投予外源性IL-12時，毒性作用已限制在治療環境中進行使用。驚人的是，本文所述之方法能夠促進內源性產生IL-12(此可在血漿中產生可全身性偵測含量之IL-12)而無毒性。此結果可實現使用患者自身免疫系統來利用因存在針對減少及/或消除腫瘤及癌細胞之IL-12而產生之益處。 Advantageously, the systemic production of endogenous IL-12 in a patient results in minimal or no toxicity to the patient. Patients may experience transient symptoms, such as transient flu-like symptoms. In general, when exogenous IL-12 has been administered to patients, the toxic effects have been limited to use in a therapeutic setting. Surprisingly, the methods described herein are capable of promoting endogenous production of IL-12 (which can produce systemically detectable levels of IL-12 in plasma) without toxicity. This result may enable the use of the patient's own immune system to take advantage of the benefits arising from the presence of IL-12 directed at reducing and / or eliminating tumors and cancer cells.

此等方法之用途亦可適用於減少及/或消除有利地對Th1環境，詳言之對IL-12起反應之其他疾病。此等疾病包含癌症；感染性疾病，包含慢性病毒及細胞內細菌或分支桿菌疾病，諸如B型肝炎、C型肝炎、HIV1、HIV2、HTLV1、HTLV2、HPV、結核分枝桿菌、牙周疾病；及過敏性疾病，如異位性氣喘。此外，促進內源性產生IL-12之方法可藉由維持細胞免疫性而具有抗衰老作用。Th1細胞與Th2細胞在正常個體中之平衡作為衰老過程之一部分而降低，使得老年人更易患感染性疾病及癌症。促進內源性IL-12產生可增加Th1/Th2比率，由此防止易患病性。 The use of these methods can also be applied to reduce and / or eliminate other diseases that respond favorably to the Th1 environment, specifically to IL-12. These diseases include cancer; infectious diseases, including chronic viruses and intracellular bacterial or mycobacterial diseases such as hepatitis B, hepatitis C, HIV1, HIV2, HTLV1, HTLV2, HPV, Mycobacterium tuberculosis, periodontal disease; And allergic diseases such as atopic asthma. In addition, methods that promote endogenous production of IL-12 can have anti-aging effects by maintaining cellular immunity. The balance of Th1 cells and Th2 cells in normal individuals is reduced as part of the aging process, making the elderly more susceptible to infectious diseases and cancer. Promoting endogenous IL-12 production can increase the Th1 / Th2 ratio, thereby preventing susceptibility to disease.

本發明之組成物通常包含外來抗原，較佳包含同種異體抗原。組成物亦包含至少一種Th1細胞激素及/或至少一種能夠誘導DC成熟以產生IL-12之DC效應分子。治療組成物通常包含至少一種Th1細胞激素及/或至少一種與同種異體抗原組合在一起之DC效應分子。組成物較佳含有能夠以單一細胞類型提供組成物之各組分之同種異體活化的活 T細胞。在較佳實施方式中，使用經活化以產生Th1細胞激素(諸如干擾素-γ、腫瘤壞死因子-α及介白素-2)且在細胞表面上表現DC成熟效應分子CD40L之同種異體Th1活細胞。或者，組成物之三種組分可源於一種以上細胞類型。 舉例而言，Th1細胞激素可源於組成物中之一種細胞類型且同種異體抗原源於分別的細胞類型且DC效應分子源於第三細胞類型。或者，一種細胞類型可含有任何兩種組分且第二細胞類型含有第三組分。細胞類型不需要為活著的，只要其提供組成物之必要組分之來源即可。 The composition of the present invention usually contains a foreign antigen, preferably an alloantigen. The composition also contains at least one Th1 cytokine and / or at least one DC effector molecule capable of inducing DC maturation to produce IL-12. Therapeutic compositions typically include at least one Th1 cytokine and / or at least one DC effector molecule combined with an alloantigen. The composition preferably contains an allogeneic activating activity capable of providing the components of the composition in a single cell type. T cells. In a preferred embodiment, an allogeneic Th1 activity that is activated to produce Th1 cytokines (such as interferon-γ, tumor necrosis factor-α, and interleukin-2) and exhibits a DC maturation effector molecule CD40L on the cell surface cell. Alternatively, the three components of the composition may be derived from more than one cell type. For example, the Th1 cytokine can be derived from one cell type in the composition and the alloantigen can be derived from a separate cell type and the DC effector molecule can be derived from a third cell type. Alternatively, one cell type may contain any two components and the second cell type contains a third component. The cell type need not be alive as long as it provides a source of the necessary components of the composition.

或者，組成物組分可源於天然或經生物工程改造之蛋白質。舉例而言，重組或純化之Th1細胞激素或DC成熟分子或同種異體抗原可一起使用或與活細胞組分組合使用。 組成物組分可在「晶片」或生物可降解平台上組合。組分不需要同時傳遞至患者，但可以任何順序傳遞。 Alternatively, the composition components may be derived from natural or bioengineered proteins. For example, recombinant or purified Th1 cytokines or DC mature molecules or alloantigens can be used together or in combination with live cell components. The composition components can be combined on a "chip" or a biodegradable platform. The components need not be delivered to the patient at the same time, but can be delivered in any order.

治療組成物中之同種異體抗原必須用以下方式提供：抗原可經吞噬或呈現至免疫系統以經處理並呈現至T細胞。抗原可為活細胞之天然部分或可使用分子生物技術加以改變或生物工程改造。抗原可為可溶的或固定在表面、活生物體或細胞之完整部分、或減毒生物體之一部分上。 在較佳實施方式中，同種異體抗原為同種異體T細胞，且在更佳實施方式中，同種異體抗原為同種異體活化的T細胞。 The alloantigen in the therapeutic composition must be provided in such a way that the antigen can be phagocytosed or presented to the immune system for processing and presentation to T cells. Antigens can be natural parts of living cells or can be altered or bioengineered using molecular biotechnology. The antigen may be soluble or immobilized on a surface, a whole part of a living organism or cell, or a part of an attenuated organism. In a preferred embodiment, the alloantigen is an allogeneic T cell, and in a more preferred embodiment, the alloantigen is an allogeneic activated T cell.

在一個例示性實施方式中，治療組成物包含在T細胞上表現之同種異體抗原。T細胞較佳為CD4+ T細胞，且更 佳為Th1細胞。Th1細胞可自源於正常血液供體之天然CD4+前驅細胞進行試管內分化、擴增及活化。較佳地，細胞在投予時呈活化狀態。較佳地，細胞由針對CD3/CD28表面分子之交聯單株抗體活化。交聯較佳藉由將CD3/CD28單株抗體固定在表面上來產生。較佳地，表面為微米或奈米(nana)珠粒粒子。珠粒可為生物可降解珠粒。此等細胞可產生大量發炎性Th1細胞激素且在細胞表面上表現用於藉由引起內源性IL-12產生來促進Th1免疫性發展之效應分子，諸如CD40L。 In an exemplary embodiment, the therapeutic composition comprises an alloantigen expressed on T cells. T cells are preferably CD4 + T cells, and more Preferably, it is a Th1 cell. Th1 cells can be differentiated, expanded and activated in vitro from natural CD4 + precursor cells derived from normal blood donors. Preferably, the cells are activated when administered. Preferably, the cells are activated by a cross-linked monoclonal antibody directed against a CD3 / CD28 surface molecule. Cross-linking is preferably produced by immobilizing a CD3 / CD28 monoclonal antibody on a surface. Preferably, the surface is micron or nano bead particles. The beads may be biodegradable beads. These cells can produce large amounts of inflammatory Th1 cytokines and appear on the cell surface to effector molecules, such as CD40L, that promote Th1 immune development by causing endogenous IL-12 production.

在較佳實施方式中，治療組成物包含活化的同種異體Th1細胞。此等活化的Th1細胞可為強力發炎劑。此等活化的同種異體Th1細胞及其製備方法例如描述於美國專利第7,435,592、7,678,572、7,402,431及7,592,431號中且以引用方式納入本文中。故意地使活化的同種異體Th1細胞與患者不匹配。 In a preferred embodiment, the therapeutic composition comprises activated allogeneic Th1 cells. These activated Th1 cells can be potent inflammatory agents. These activated allogeneic Th1 cells and methods of making them are described, for example, in U.S. Patent Nos. 7,435,592, 7,678,572, 7,402,431, and 7,592,431 and are incorporated herein by reference. The activated allogeneic Th1 cells were intentionally mismatched with the patient.

多種Th1發炎性細胞激素可包含在治療組成物中。發炎性Th1細胞激素之實例包含：IL-1、IL-2、IL-6、IL-12、IL-15、IFN-γ、TNF-α、TNF-β、GM-CSF及C-C趨化素且不包含TGF-β、IL-4或IL-10。細胞激素組分可為天然或重組細胞激素或可為設計來與細胞激素之受體相互作用之經生物工程改造的分子。細胞激素可直接包含在治療組成物中。或者，治療組成物可包含活細胞或產生並分泌細胞激素之其他組分。較佳地，因為外源性細胞激素傾向於對患者具有極大毒性而內源性細胞激素並非如此，所以細胞激 素係經由活化的細胞來源天然提供。在一些例示性實施方式中，治療組成物包含呈活化狀態之T細胞，其正在產生並分泌發炎性Th1細胞激素，因此可充當治療組成物中此等細胞激素之來源。 A variety of Th1 inflammatory cytokines can be included in the therapeutic composition. Examples of inflammatory Th1 cytokines include: IL-1, IL-2, IL-6, IL-12, IL-15, IFN-γ, TNF-α, TNF-β, GM-CSF and CC chemokine and Does not contain TGF-β, IL-4 or IL-10. The cytokine component can be a natural or recombinant cytokine or a bioengineered molecule that can be designed to interact with a receptor for a cytokine. Cytokines can be included directly in the therapeutic composition. Alternatively, the therapeutic composition may comprise living cells or other components that produce and secrete cytokines. Preferably, because exogenous cytokines tend to be extremely toxic to patients and endogenous cytokines are not, cytokine The vegetative line is provided naturally via an activated cell source. In some exemplary embodiments, the therapeutic composition comprises T cells in an activated state, which is producing and secreting inflammatory Th1 cytokines, and thus may serve as a source of these cytokines in the therapeutic composition.

治療組成物可包含使得不成熟DC成熟之一或多種因子。詳言之，為促進DC1細胞成熟及IL-12產生，從而引起干擾素-γ產生及Th1繼承性免疫性之成熟因子。DC能夠自不成熟抗原捕捉細胞進化成成熟抗原呈現T細胞-將抗原轉化成免疫原且表現為引發免疫反應所必需之細胞激素、趨化素、共刺激分子之預致敏細胞(priming cell)。所誘導的由T細胞介導之免疫反應(Th1相對於Th2)之類型視自周圍微環境接收之活化信號而變化。DC調節免疫性(諸如抗腫瘤及抗感染性疾病免疫性)之能力取決於促進Th1免疫性之DC成熟化。人類DC不為同源群體。除誘導抗腫瘤免疫性之外，DC亦可誘導無因變性或耐受性。DC源於CD34+造血幹細胞(HSC)。骨髓樹突細胞(DC1)及漿細胞樣DC(DC2)為人類DC之兩種主要亞族群且其在表型、遷移及功能方面之特徵極其不同。DC1細胞為有效T細胞刺激物，誘導腫瘤特異性免疫反應。CD11c+DC1細胞主要誘導Th1分化，而表現IL-3之受體(CD123)之DC2細胞主要促進Th2反應。癌症患者中之兩種DC群體皆顯著低於健康供體中。在成熟後，DC1細胞產生IL-12且DC2細胞產生IL-10。 The therapeutic composition may comprise one or more factors that allow immature DCs to mature. In detail, in order to promote the maturation of DC1 cells and the production of IL-12, thereby causing the production of interferon-γ and the maturation factor of Th1 inherited immunity. DC can evolve from immature antigen-capturing cells to mature antigen-presenting T cells-Priming cells that convert antigens into immunogens and act as cytokines, chemokines, and costimulatory molecules necessary to trigger an immune response . The type of T cell-mediated immune response (Th1 versus Th2) induced varies depending on the activation signals received from the surrounding microenvironment. The ability of DCs to modulate immunity (such as anti-tumor and anti-infectious disease immunity) depends on DC maturation that promotes Th1 immunity. Human DCs are not homologous populations. In addition to inducing antitumor immunity, DCs can also induce non-cause degeneration or tolerance. DC is derived from CD34 + hematopoietic stem cells (HSC). Bone marrow dendritic cells (DC1) and plasma cell-like DCs (DC2) are the two main subpopulations of human DC and have extremely different phenotype, migration, and functional characteristics. DC1 cells are effective T cell stimulators and induce tumor-specific immune responses. CD11c + DC1 cells mainly induce Th1 differentiation, while DC2 cells expressing IL-3 receptor (CD123) mainly promote Th2 response. Both DC populations in cancer patients were significantly lower than in healthy donors. After maturation, DC1 cells produce IL-12 and DC2 cells produce IL-10.

在DC成熟過程期間產生諸如IL-10及IL-12之細胞激 素會影響Th1或Th2免疫反應之DC誘導。除表現高含量之抗原呈現分子及共刺激分子之外，成熟DC亦必須釋放大量IL-12以刺激Th1免疫反應。釋放IL-10會藉由干擾共刺激分子之上調及IL-12之產生，隨後限制DC引發Th1反應之能力來阻斷DC成熟過程。 Cytokines such as IL-10 and IL-12 are produced during DC maturation Affects DC induction of Th1 or Th2 immune responses. In addition to showing high levels of antigen presenting molecules and costimulatory molecules, mature DCs must also release large amounts of IL-12 to stimulate the Th1 immune response. Release of IL-10 will block DC maturation by interfering with the up-regulation of co-stimulatory molecules and the production of IL-12, and then limiting the ability of DCs to trigger a Th1 response.

多種因素可誘導DC成熟以變成DC1(即在抗原攝取及處理之後產生IL-12之細胞)，該等因素包含：完整細菌或細菌源性抗原(例如脂多醣，LPS)、發炎性細胞激素(諸如IFN-γ、TNF-α、IL-1、GM-CSF)、連接選擇細胞表面受體(例如CD40)、病毒產物(例如雙股RNA)、Fas接合於不成熟DC上，例如誘導IL-1β及IFN-γ之成熟與釋放。 連接CD40會促進上調共刺激分子B7-1/CD80及B7-2/CD86及IL-12分泌以及趨化素(例如IL-8、MIP-1α、MIP-1β)釋放。 Various factors can induce DC to mature to become DC1 (ie, cells that produce IL-12 after antigen uptake and processing). These factors include: intact bacteria or bacterial-derived antigens (such as lipopolysaccharide, LPS), inflammatory cytokines ( (Such as IFN-γ, TNF-α, IL-1, GM-CSF), ligation select cell surface receptors (e.g., CD40), viral products (e.g., double-stranded RNA), Fas conjugation to immature DCs, such as inducing Maturation and release of 1β and IFN-γ. Linking CD40 promotes up-regulation of co-stimulatory molecules B7-1 / CD80 and B7-2 / CD86 and IL-12 secretion and release of chemokines (such as IL-8, MIP-1α, MIP-1β).

在一些較佳實施方式中，包含CD40L作為用於DC成熟之因子。包含使得DC成熟之其他因子亦在本發明之範疇內。在一些例示性實施方式中，治療組成物包含在表面上表現高密度CD40L之呈活化狀態之T細胞。CD40L為一種用於DC成熟以產生IL-12之強力效應分子。 In some preferred embodiments, CD40L is included as a factor for DC maturation. It is within the scope of the invention to include other factors that allow DCs to mature. In some exemplary embodiments, the therapeutic composition comprises T cells in an activated state that exhibit high density CD40L on the surface. CD40L is a potent effector molecule for DC maturation to produce IL-12.

在一個例示性實施方式中，治療組成物包含活化的同種異體T細胞、至少一種I型細胞激素及至少一種使得DC成熟之因子。包含此等組分之組成物例如描述於2010年12月14日申請且以引用方式納入本文中之申請中美國專利申請案第12/967,910號中。 In an exemplary embodiment, the therapeutic composition comprises activated allogeneic T cells, at least one type I cytokine, and at least one factor that makes DC mature. Compositions containing these components are described, for example, in U.S. Patent Application No. 12 / 967,910, filed on Dec. 14, 2010 and incorporated herein by reference.

在消融一些腫瘤細胞之後腫瘤內投予治療組成物以使腫瘤相關之抗原釋放入微環境中可提供使DC成熟為DC1表型之強力輔助作用，該DC1表型產生IL-12且促進1型抗腫瘤免疫性顯現及腫瘤免疫逃避機制下調。治療組成物之投予亦可藉由其他方法達成，包括例如靜脈內、皮內、脊椎內(intrathecal)、腹膜內、病灶內、胸膜內投予及其類似方法。較佳地，因為皮膚富含稱為蘭格漢氏細胞(Langcrhans cell)之不成熟DC，所以組成物首先經皮內投予。在諸如干擾素-γ、腫瘤壞死因子-α、IL-2及GM-CSF之發炎性Th1細胞激素及諸如CD40L之DC成熟因子存在下，蘭格漢氏細胞攝取同種異體抗原且成熟為DC1，即產生IL-12之細胞。此等成熟細胞遷移至淋巴結且促進Th1免疫性發展。 After ablating some tumor cells, administration of a therapeutic composition intratumorally to release tumor-associated antigens into the microenvironment can provide a powerful adjuvant that matures DC into the DC1 phenotype, which produces IL-12 and promotes type 1 resistance. Tumor immune manifestations and tumor immune evasion mechanisms are down-regulated. Administration of the therapeutic composition can also be achieved by other methods, including, for example, intravenous, intradermal, intrathecal, intraperitoneal, intralesional, intrapleural administration, and the like. Preferably, because the skin is rich in immature DCs called Langcrhans cells, the composition is first administered intradermally. In the presence of inflammatory Th1 cytokines such as interferon-γ, tumor necrosis factor-α, IL-2 and GM-CSF, and DC maturation factors such as CD40L, Langerhans cells take up alloantigens and mature to DC1, Cells that produce IL-12. These mature cells migrate to the lymph nodes and promote Th1 immune development.

皮內注射組成物可使患者「預致敏(prime)」以變得對組成物中之同種異體抗原具有免疫性。多次皮內注射可增加對患者循環中同種異體抗原具有特異性之Th1記憶細胞之數目，此又會改變Th1/Th2平衡。注射1×10⁶個細胞至1×10⁷個同種異體活化的Th1細胞為較佳皮內劑量，1ml流體中有1×10⁷個細胞為最佳的。較佳重複皮內給藥多次以增進循環Th1記憶細胞之數目。皮內給藥頻率較佳為每7天注射約3-4次，更佳每3-4天注射約3-4次。 Intradermal injections of the composition can "prime" the patient to become immune to the alloantigen in the composition. Multiple intradermal injections can increase the number of Th1 memory cells that are specific to the alloantigen in the patient's circulation, which in turn changes the Th1 / Th2 balance. Injecting 1 × 10 ⁶ cells to 1 × 10 ⁷ allo-activated Th1 cells is the preferred intradermal dose, and 1 × 10 ⁷ cells in 1 ml of fluid is the best. The intradermal administration is preferably repeated multiple times to increase the number of circulating Th1 memory cells. The frequency of intradermal administration is preferably about 3-4 injections every 7 days, and more preferably about 3-4 injections every 3-4 days.

在較佳實施方式中，皮內給藥之後進行組成物之腫瘤內給藥以產生原位疫苗。較佳在原位消融目標病灶中的一些腫瘤細胞之後進行腫瘤內給藥。消融較佳藉由使用極冷 (低溫消融)或極熱(輻射)來達成，但亦可使用多種方法，包括酒精消融、化學療法及/或單株抗體藥物來進行。 較佳腫瘤內劑量介於約1×10⁷個細胞與1×10⁸個細胞之間，最佳為約3×10⁷個細胞。較佳的是，第一腫瘤內劑量係在消融之後即刻注射且第二腫瘤內劑量係在第一次注射之後約7天內、較佳約3-4天內注射。消融之後進行腫瘤內注射組成物之此方法必要時可重複。 In a preferred embodiment, the composition is administered intratumorally after intradermal administration to produce an in situ vaccine. The intratumoral administration is preferably performed after ablating some tumor cells in the target lesion in situ. Ablation is preferably achieved by using extremely cold (low temperature ablation) or extreme heat (radiation), but multiple methods can also be used, including alcohol ablation, chemotherapy, and / or monoclonal antibody drugs. The preferred intratumoral dose is between about 1 × 10 ⁷ cells and 1 × 10 ⁸ cells, and most preferably about 3 × 10 ⁷ cells. Preferably, the first intratumoral dose is injected immediately after ablation and the second intratumoral dose is injected about 7 days, preferably about 3-4 days after the first injection. This method of injecting the composition after tumor ablation can be repeated if necessary.

方法亦較佳包括靜脈內投予組成物以使宿主免疫細胞(先天性與繼承性兩者)活化及其外滲至發炎部位，包含腫瘤位置。同種異體活化的Th1細胞之組成物之靜脈內劑量較佳包含約1×10⁷至1×10⁹個細胞、更佳約5×10⁷至1×10⁸個細胞。靜脈內輸注可較佳按月重複數次。 The method also preferably includes intravenously administering the composition to activate host immune cells (both congenital and inherited) and extravasate to the site of inflammation, including the tumor site. The intravenous dose of a composition of allo-activated Th1 cells preferably comprises about 1 × 10 ⁷ to 1 × 10 ⁹ cells, more preferably about 5 × 10 ⁷ to 1 × 10 ⁸ cells. Intravenous infusions are preferably repeated several times a month.

組成物之同種異體Th1細胞較佳產生大量1型細胞激素：IL2、IFN-γ、TNF-α及GM-CSF。在不成熟DC吞噬及處理抗原所處之微環境中存在發炎性Th1細胞激素可有助於促進成熟為DC1，即產生IL-12之DC。IL-12可刺激IFN-γ之含量，此又可引起促進Th1免疫性。IFN-γ為一種為促進1型抗腫瘤免疫性所必需之關鍵1型細胞激素。IFN-γ可藉由直接抑制腫瘤細胞生長及誘導T細胞介導之抗腫瘤反應來介導抗腫瘤作用。IFN-γ分泌可獨立地促成NK細胞反應且增強由IL-12活化之NK細胞反應。 The allogeneic Th1 cells of the composition preferably produce a large number of type 1 cytokines: IL2, IFN-γ, TNF-α, and GM-CSF. The presence of inflammatory Th1 cytokines in the microenvironment where immature DCs are phagocytosing and processing antigens can help promote maturation to DC1, the DC that produces IL-12. IL-12 can stimulate the content of IFN-γ, which in turn can promote the promotion of Th1 immunity. IFN-γ is a key type 1 cytokine necessary to promote type 1 antitumor immunity. IFN-γ can mediate antitumor effects by directly inhibiting tumor cell growth and inducing T cell-mediated antitumor responses. IFN-γ secretion can independently contribute to NK cell responses and enhance NK cell responses activated by IL-12.

含有活化的同種異體Th1細胞之較佳醫藥品可源於自正常經篩檢血液供體純化之前驅體。細胞應以經調配用於皮內或腫瘤內注射或靜脈內輸注之無菌低內毒素劑型供 應。細胞亦可經調配用於腹膜內、胸膜內或硬膜外輸注。 供體較佳經測試為HIV1、HIV2、HTLV1、HTLV2、HBV、HCV、RPR(梅毒)陰性，且細胞較佳經測試為黴漿菌(mycoplasma)、EBV及CMV陰性。在較佳實施方式中，活化的同種異體細胞為與患者不匹配之HLA。 Preferred medicines containing activated allogeneic Th1 cells may be derived from a precursor purified from a normal screened blood donor. Cells should be supplied in sterile low endotoxin dosage forms formulated for intradermal or intratumoral injection or intravenous infusion. should. Cells can also be formulated for intraperitoneal, intrapleural or epidural infusion. The donor is preferably tested negative for HIV1, HIV2, HTLV1, HTLV2, HBV, HCV, RPR (syphilis), and the cells are preferably tested negative for mycoplasma, EBV and CMV. In a preferred embodiment, the activated allogeneic cells are HLAs that do not match the patient.

本發明方法大體上係關於在患者血漿中產生可偵測含量之內源性IL-12。方法包括以工程改造患者免疫系統以在患者血漿中產生可偵測含量之內源性IL-12之方式來投予本發明組成物。本文所述之方法可增加癌症患者體內Th1免疫細胞之循環數目，從而使平衡自Th2環境移至Th1環境。另外，方法亦可包括以下步驟：引發抗腫瘤特異性Th1免疫性及/或活化先天性及繼承性免疫反應之組分以產生持續Th1細胞激素環境來下調腫瘤免疫逃避。 The methods of the present invention are generally directed to producing detectable levels of endogenous IL-12 in a patient's plasma. The method includes administering a composition of the present invention by engineering a patient's immune system to produce a detectable amount of endogenous IL-12 in the patient's plasma. The methods described herein can increase the number of Th1 immune cells circulating in cancer patients, thereby shifting the balance from the Th2 environment to the Th1 environment. In addition, the method may include the steps of: triggering anti-tumor-specific Th1 immunity and / or activating components of the innate and inherited immune response to generate a sustained Th1 cytokine environment to downregulate tumor immune escape.

本發明方法可包括投予含有外來抗原之組成物以促進患者針對該外來抗原之Th1免疫性。方法亦可包括消融腫瘤之全部或一部分，此使得至少一些腫瘤壞死。多種方法可用於在患者中產生腫瘤壞死。方法亦可涉及在腫瘤壞死部位，亦即腫瘤病灶部位附近創建發炎性微環境。此外，方法亦可包括活化患者之繼承性及先天性免疫細胞以維持延長之Th1環境。在較佳實施方式中，方法之關鍵部分包括使用含有如上所述之活化的同種異體T細胞之醫藥品或組成物。 The method of the present invention may include administering a composition containing a foreign antigen to promote a patient's Th1 immunity against the foreign antigen. The method may also include ablating all or a portion of the tumor, which necroses at least some of the tumor. A variety of methods can be used to produce tumor necrosis in a patient. The method may also involve creating an inflammatory microenvironment near the tumor necrosis site, that is, near the tumor lesion site. In addition, the method may include activating the patient's inherited and innate immune cells to maintain a prolonged Th1 environment. In a preferred embodiment, a key part of the method involves the use of a pharmaceutical or composition containing activated allogeneic T cells as described above.

因為大多數人類癌症患者通常呈現有極化Th2免疫性，所以此治療方法之目標大體上在於增加癌症患者體內 循環Th1細胞之量。可藉由向患者投予一種包含外來抗原之上述治療組成物在癌症患者體內增進循環Th1細胞之數目。 Because most human cancer patients usually exhibit polarized Th2 immunity, the goal of this treatment is to increase the body of cancer patients. Circulate the amount of Th1 cells. The number of circulating Th1 cells can be increased in a cancer patient by administering the above-mentioned therapeutic composition containing a foreign antigen to the patient.

在一個例示性實施方式中，向患者投予皮內注射之活化的同種異體Th1細胞。在較佳實施方式中，皮內注射係根據每週時程，一週一次，持續約3-4週。在其他較佳實施方式中，皮內注射可約每3-4天投予多次。皮內注射可每兩天或至多相隔一年加以投予。注射時程應係設計來增強Th1記憶細胞在循環中的足跡(footprint)。在外來細胞上表現之同種異體抗原可刺激強力免疫排斥反應。此外，Th1細胞激素存在於組成物中或由同種異體細胞表現Th1細胞激素可提供為引導針對同種異體抗原之免疫反應朝向Th1記憶免疫性所必需之發炎性輔助環境。此可在患者體內之循環中產生增加之Th1記憶細胞池，該等Th1記憶細胞對同種異體Th1細胞內所含之同種異體抗原具有特異性。多次投藥可充當增效(booster)注射，從而增加對同種異體抗原具有特異性之循環記憶Th1細胞之數目。 In an exemplary embodiment, a patient is administered an activated allogeneic Th1 cell injected intradermally. In a preferred embodiment, the intradermal injection is based on a weekly schedule, once a week for about 3-4 weeks. In other preferred embodiments, the intradermal injection may be administered multiple times about every 3-4 days. Intradermal injections can be administered every two days or at most one year apart. The duration of the injection should be designed to enhance the footprint of the Th1 memory cells in the circulation. Alloantigens expressed on foreign cells can stimulate a strong immune rejection. In addition, Th1 cytokines are present in the composition or expressed by allogeneic cells. Th1 cytokines can provide the inflammatory auxiliary environment necessary to direct the immune response against alloantigens towards Th1 memory immunity. This can generate an increased pool of Th1 memory cells in the circulation in the patient's body, which are specific for the alloantigen contained in the allogeneic Th1 cells. Multiple administrations can serve as booster injections, thereby increasing the number of circulating memory Th1 cells specific for alloantigens.

在一些實施方式中，投予同種異體活化的T細胞之後可進行其他步驟以增強患者反應。此等步驟可包含例如消融腫瘤(此會導致腫瘤壞死)以及腫瘤內投予其他同種異體活化的T細胞。亦可進行其他靜脈內投予同種異體活化的細胞。此等方法描述於Har-Noy之美國專利第7,972,594號中，該專利係以引用方式納入本文中。 In some embodiments, additional steps can be performed after administration of allo-activated T cells to enhance patient response. These steps may include, for example, ablation of the tumor (which causes tumor necrosis) and intratumoral administration of other allogeneic activated T cells. Other allogeneic activated cells can also be administered intravenously. Such methods are described in Har-Noy, US Patent No. 7,972,594, which is incorporated herein by reference.

使用本文所述之方法投予治療組成物或醫藥品可促進 在患者體內由患者自身免疫系統全身性產生內源性IL-12。 患者體內的內源性IL-12之濃度為足夠的，以致可在患者血漿中偵測到IL-12。因為組成物之組分通常在藉由投予同種異體物質所引發之排斥反應中由患者之免疫系統消除，所以可偵測含量之IL-12為內源性的而非可能存在於治療組成物中之任何IL-12之結果。在較佳實施方式中，組成物含有不能產生IL-12之T細胞。因此，在患者血漿中偵測到之任何IL-12皆為由患者自身免疫系統產生之IL-12之結果。 Administration of a therapeutic composition or pharmaceutical using the methods described herein can promote Endogenous IL-12 is produced systemically by the patient's own immune system. The endogenous IL-12 concentration in the patient is sufficient so that IL-12 can be detected in the patient's plasma. Because the components of the composition are usually eliminated by the patient's immune system in a rejection response induced by the administration of an allogeneic substance, the detectable amount of IL-12 is endogenous and may not be present in the therapeutic composition Any IL-12 results. In a preferred embodiment, the composition contains T cells that are not capable of producing IL-12. Therefore, any IL-12 detected in the patient's plasma is the result of IL-12 produced by the patient's own immune system.

較佳地，IL-12係由患者之免疫細胞，例如患者之自身單核球、自然殺手細胞及樹突細胞產生。此等細胞將在藉由投予本文所述之組成物所產生之發炎性細胞激素或I型細胞激素的影響下成熟。 Preferably, IL-12 is produced by a patient's immune cells, such as the patient's own monocytes, natural killer cells, and dendritic cells. These cells will mature under the influence of inflammatory cytokines or type I cytokines produced by administration of the compositions described herein.

患者血漿中IL-12之濃度可變化但通常為至少約8000pg/ml。患者血漿中IL-12之濃度較佳介於約8000pg/ml至200,000pg/ml之間。如本文所述，在患者血漿中偵測到之IL-12濃度不導致毒性問題。然而，已知投予外源性IL-12對患者具有毒性。相較於在其血清中不表現IL-12之患者，血清轉化成血漿中IL-12表現之患者之存活率增加。IL-12之含量可能不與存活率相關，僅IL-12之存在為關鍵的。 The IL-12 concentration in the patient's plasma can vary but is usually at least about 8000 pg / ml. The IL-12 concentration in the patient's plasma is preferably between about 8000 pg / ml and 200,000 pg / ml. As described herein, IL-12 concentrations detected in patient plasma do not cause toxicity issues. However, administration of exogenous IL-12 is known to be toxic to patients. Survival of patients with serum-expressed IL-12 expression was increased compared to patients with no IL-12 expression in their serum. The content of IL-12 may not be related to survival rate, only the presence of IL-12 is critical.

通常在投予組成物之後，在一段時期之後偵測到IL-12增加。較佳地，在用治療組成物給藥約3-4週之後，可在血漿中偵測到IL-12。IL-12血清轉化可存在延遲，投予末次組成物之後的約90-120天。 An increase in IL-12 is usually detected after a period of time after the composition is administered. Preferably, IL-12 is detectable in plasma after administration of the therapeutic composition for about 3-4 weeks. There may be a delay in IL-12 seroconversion, about 90-120 days after administration of the last composition.

血漿中的IL-12可藉由使用多種方法偵測。IL-12具有 兩個稱為p40及p35鏈之次單元且對p40具有特異性之抗體較佳用於偵測。若干方法可用於偵測IL-12之存在。IL-12之偵測可包含例如ELISA及細胞激素珠粒陣列。 IL-12 in plasma can be detected by using a variety of methods. IL-12 has Two subunits called p40 and p35 chains and specific for p40 are preferred for detection. Several methods can be used to detect the presence of IL-12. Detection of IL-12 may include, for example, ELISA and cytokine bead arrays.

本文所述之方法可適於多種患者，包含人類。方法亦可對其他哺乳動物使用。 The methods described herein can be adapted to a variety of patients, including humans. The method can also be applied to other mammals.

本發明亦包含治療患者疾病之方法。疾病可包含如上所述之癌性腫瘤、血液學惡性腫瘤以及由病原體媒介引起之疾病。患者之對Th1反應敏感之其他疾病亦可使用本文所述之方法治療。根據本文所述之方法向患者投予同種異體組成物。接著監測患者血漿之IL-12存在。偵測內源性IL-12可指示患者對疾病之免疫反應。可進行治療組成物之其他投予以維持IL-12含量且藉此維持患者針對疾病抗原之免疫反應。 The invention also includes methods for treating a patient's disease. Diseases can include cancerous tumors, hematological malignancies, and diseases caused by pathogen vectors, as described above. Other diseases in which a patient is susceptible to Th1 can also be treated using the methods described herein. The allogeneic composition is administered to a patient according to the methods described herein. The patient's plasma was then monitored for the presence of IL-12. Detection of endogenous IL-12 can indicate a patient's immune response to the disease. Other administrations of the therapeutic composition may be performed to maintain the IL-12 content and thereby maintain the patient's immune response against the disease antigen.

實施例 Examples

進行此研究以監測用同種異體活化的Th1細胞治療之患者的血漿中的IL-12含量。此等活化的同種異體Th1細胞及其製備方法描述於美國專利第7,435,592號中。故意地使活化的同種異體Th1細胞與患者不匹配。 This study was performed to monitor the IL-12 content in the plasma of patients treated with allo-activated Th1 cells. These activated allogeneic Th1 cells and methods of making them are described in US Patent No. 7,435,592. The activated allogeneic Th1 cells were intentionally mismatched with the patient.

皮內注射-投予活化的同種異體Th1細胞之皮內注射液。將細胞在1×10⁷個細胞/毫升之密度下懸浮於1ml中。 Intradermal injection -An intradermal injection of activated allogeneic Th1 cells. The cells were suspended in 1 ml at a density of 1 × 10 ⁷ cells / ml.

腫瘤內注射-在消融一小時內在經消融腫瘤之壞死中心投予腫瘤內注射液。 Intratumoral injection -Intratumoral injection is administered at the ablation tumor necrosis center within one hour of ablation.

使用CryoCare-28經皮探針系統(CryoCare-28 Percutaneous Probe System)(Endocare,CA,USA)進行低溫消融。此系統使用焦耳-湯姆遜效應(Joule-Thomson effect)來冷卻封閉系統中低溫探針之末端。根據氣體係數及噴嘴尺寸，不同氣體要素在接近噴嘴之區域處產生不同熱交換事件。氬氣用於冷卻(-187℃)，且氦氣用於加熱(67℃)。 CryoCare-28 Percutaneous Probe System Probe System) (Endocare, CA, USA). This system uses the Joule-Thomson effect to cool the ends of cryogenic probes in a closed system. According to the gas coefficient and the nozzle size, different gas elements generate different heat exchange events in the area close to the nozzle. Argon was used for cooling (-187 ° C) and helium was used for heating (67 ° C).

鑒別計劃之目標腫瘤病灶且在CT影像引導下定位。產生無菌場且向計劃之探針***部位投予局部麻醉。經皮***引導探針且藉由CT驗證在目標腫瘤病灶內。進行一或兩個凍融循環。根據目標腫瘤之尺寸使用2mm或5mm之單一探針。視達成「冰球」在CT上可見而定，冷凍時間為約5-20分鐘。藉由在等於冷凍時間之階段期間饋入氦氣來達成解凍，隨後起始第二冷凍製程。程序要求消融腫瘤病灶之樣品且不要求與無腫瘤邊緣之完全消融腫瘤。 Identify the planned target tumor lesions and locate them under the guidance of CT images. A sterile field is generated and local anesthesia is administered to the planned probe insertion site. A guide probe is inserted percutaneously and verified by CT within the target tumor lesion. Perform one or two freeze-thaw cycles. Use a single probe of 2mm or 5mm depending on the size of the target tumor. Depending on the "ice hockey" visible on the CT, the freezing time is about 5-20 minutes. Thawing is achieved by feeding helium during a phase equal to the freezing time, and then a second freezing process is initiated. The procedure requires ablation of a tumor lesion sample and does not require complete ablation of the tumor with no tumor margin.

在第二冷凍循環之後使病灶冷卻，隨後注射同種異體活化的Th1細胞。 After the second freezing cycle, the lesions were cooled and subsequently injected with allo-activated Th1 cells.

表1、2及3展示特定治療之時序及在指定日患者血漿中的IL-12含量。圖1為說明在研究期間由患者免疫系統達成之IL-12表現之圖。 Tables 1, 2 and 3 show the timing of specific treatments and IL-12 levels in the plasma of patients on a given day. Figure 1 is a graph illustrating IL-12 performance achieved by the patient's immune system during the study.

儘管本發明已參考較佳實施方式加以描述，但熟習該項技術者將認識到可在不脫離本發明之精神及範疇下在形式及細節方面作出變化。 Although the present invention has been described with reference to preferred embodiments, workers skilled in the art will recognize that changes may be made in form and detail without departing from the spirit and scope of the invention.

圖1為說明歷經超過1年患者(患者#11)血漿中IL-12含量之圖。使用各種投予模式在各個時間向患者投予同種異體活化的Th-1細胞。 Figure 1 is a graph illustrating the IL-12 content in the plasma of a patient (patient # 11) after more than one year. Allogeneously activated Th-1 cells are administered to patients at various times using various administration modes.

Claims (15)

一種組成物的用途，其係用於製造用於治療癌症及/或感染性疾病的醫藥品，其中該組成物包含活化的同種異體Th1細胞，以及表現在該等Th1細胞表面上的樹突細胞成熟分子CD40L及/或FasL，其中該醫藥品係用於包含以下步驟的方法中：將該醫藥品以多劑形式投予以在投予最後的該醫藥品後約90天之後在人類患者血漿中提供至少8000pg/ml的內源性IL-12，以及監測該人類患者血漿中內源性IL-12之存在。Use of a composition for the manufacture of a medicament for treating cancer and / or infectious diseases, wherein the composition comprises activated allogeneic Th1 cells and dendritic cells expressed on the surface of the Th1 cells The mature molecule CD40L and / or FasL, wherein the pharmaceutical product is used in a method comprising the step of: administering the pharmaceutical product in multiple doses in human plasma after about 90 days after the final administration Provide at least 8000 pg / ml of endogenous IL-12, and monitor the presence of endogenous IL-12 in the plasma of the human patient. 如申請專利範圍第1項之用途，其中該組成物進一步包含以下一或多者：IL-1、IL-2、IL-6、IL-8、IL-15、干擾素-γ、TNF-α、GM-CSF。For example, the application of the scope of patent application, wherein the composition further comprises one or more of the following: IL-1, IL-2, IL-6, IL-8, IL-15, interferon-γ, TNF-α GM-CSF. 如申請專利範圍第1或2項之用途，其中該等Th1細胞藉由交聯CD3與CD28活化。For example, the application of the scope of patent application item 1 or 2, wherein the Th1 cells are activated by cross-linking CD3 and CD28. 如申請專利範圍第3項之用途，其中該組成物進一步包含以下Th1細胞激素中的一或多者：IL-2、IFN-γ及GM-CSF。For example, the application in the scope of patent application No. 3, wherein the composition further comprises one or more of the following Th1 cytokines: IL-2, IFN-γ, and GM-CSF. 如申請專利範圍第1或2項之用途，其中該組成物之組分被固定在表面上。For example, the application in the scope of patent application No. 1 or 2, wherein the components of the composition are fixed on the surface. 如申請專利範圍第5項之用途，其中該表面為生物可降解的。Such as the application of the scope of patent application No. 5, wherein the surface is biodegradable. 如申請專利範圍第1或2項之用途，其中該等Th1細胞被包裝在注射器或可撓性容器中。In the case of applying for the application of item 1 or 2, the Th1 cells are packed in a syringe or a flexible container. 如申請專利範圍第7項之用途，其中該等細胞之濃度為1×10⁷個細胞/毫升或1×10⁷個細胞/毫升以上。The patent application 242,800 range of 7, wherein the concentration of the cells was 1 × 10 ⁷ cells / ml or 1 × 10 ⁷ cells / ml or more. 如申請專利範圍第8項之用途，其中該等細胞被懸浮於無養分培養基中。For the purpose of applying for the scope of patent No. 8, wherein the cells are suspended in a nutrient-free medium. 如申請專利範圍第1項之用途，其中所述在患者血漿中之內源性IL-12的量對該患者無毒性。The use as claimed in the scope of patent application item 1, wherein the amount of endogenous IL-12 in the patient's plasma is non-toxic to the patient. 如申請專利範圍第1項之用途，其中該等Th1細胞藉由交聯一或多種結合於該等Th1細胞上之細胞表面部分之媒介而活化。For example, the application in the scope of patent application, wherein the Th1 cells are activated by cross-linking one or more mediators that bind to the cell surface portion of the Th1 cells. 如申請專利範圍第11項之用途，其中該一或多種媒介為單株抗體。For example, the application in the scope of patent application No. 11 wherein the one or more mediators are monoclonal antibodies. 如申請專利範圍第12項之用途，其中該等單株抗體為抗CD3及抗CD28單株抗體。Such as the application of the scope of application of the patent No. 12, wherein the monoclonal antibodies are anti-CD3 and anti-CD28 monoclonal antibodies. 如申請專利範圍第1、2及10-13項中任一項之用途，其中該醫藥品以不小於每3天1次的頻率投予。For example, the application of any one of items 1, 2 and 10-13, wherein the medicine is administered at a frequency of not less than once every 3 days. 如申請專利範圍第1、2及10-13項中任一項之用途，其中該醫藥品是藉由皮內、靜脈內或病灶內途徑投予。For example, the application of any one of claims 1, 2 and 10-13, wherein the medicine is administered by intradermal, intravenous or intralesional route.